Toll-Like Receptor 3 Antagonists

ABSTRACT

Toll Like Receptor 3 (TLR3) antibody antagonists, polynucleotides encoding TLR3 antibody antagonists or fragments thereof, and methods of making and using the foregoing are disclosed.

CROSS-REFERENCE TO RELATED APPLICATIONS

This application is a divisional of U.S. application Ser. No.13/973,187, filed 22 Aug. 2013, currently allowed, which is a divisionalof U.S. application Ser. No. 12/770,147, filed 29 Apr. 2010, now U.S.Pat. No. 8,540,986, which is a continuation-in-part of U.S. applicationSer. No. 12/609,675, filed 30 Oct. 2009, now U.S. Pat. No. 8,409,567,which claims the benefit of U.S. Provisional Application No. 61/109,974,filed 31 Oct. 2008 and U.S. Provisional Application No. 61/161,860,filed 20 Mar. 2009 and U.S. Provisional Application No. 61/165,100,filed 31 Mar. 2009 and U.S. Provisional Application No. 61/173,686,filed 29 Apr. 2009, the entire contents of which are incorporated hereinby reference.

FIELD OF THE INVENTION

The present invention relates to Toll-Like Receptor 3 (TLR3) antibodyantagonists, polynucleotides encoding TLR3 antibody antagonists orfragments thereof, and methods of making and using the foregoing.

BACKGROUND OF THE INVENTION

Toll-like receptors (TLRs) regulate activation of the innate immuneresponse and influence the development of adaptive immunity byinitiating signal transduction cascades in response to bacterial, viral,parasitic, and in some cases, host-derived ligands (Lancaster et al., J.Physiol. 563:945-955, 2005). The plasma membrane localized TLRs, TLR1,TLR2, TLR4 and TLR6 recognize ligands including protein or lipidcomponents of bacteria and fungi. The predominantly intracellular TLRs,TLR3, TLR7 and TLR9 respond to dsRNA, ssRNA and unmethylated CpG DNA,respectively. Dysregulation of TLR signaling is believed to cause amultitude of problems, and therapeutic strategies are in developmenttowards this axis (Hoffman et al., Nat. Rev. Drug Discov. 4:879-880,2005; Rezaei, Int. Immunopharmacol. 6:863-869, 2006; Wickelgren, Science312:184-187, 2006). For example, antagonists of TLR4 and TLRs 7 and 9are in clinical development for severe sepsis and lupus, respectively(Kanzler et al., Nat. Med. 13:552-559, 2007).

TLR3 signaling is activated by dsRNA, mRNA or RNA released from necroticcells during inflammation or virus infection. TLR3 activation inducessecretion of interferons and pro-inflammatory cytokines and triggersimmune cell activation and recruitement that are protective duringcertain microbial infections. For example, a dominant-negative TLR3allele has been associated with increased susceptibility to HerpesSimplex encephalitis upon primary infection with HSV-1 in childhood(Zheng et al., Science 317:1522-1527, 2007). In mice, TLR3 deficiency isassociated with decreased survival upon coxsackie virus challenge(Richer et al., PLoS One 4:e4127, 2009). However, uncontrolled ordysregulated TLR3 signaling has been shown to contribute to morbidityand mortality in certain viral infection models including West Nile,phlebovirus, vaccinia, and influenza A (Wang et al., Nat. Med.10:1366-1373, 2004; Gowen et al., J. Immunol. 177:6301-6307, 2006;Hutchens et al., J. Immunol. 180:483-491, 2008; Le Goffic et al., PloSPathog. 2:E53, 2006).

The crystal structures of the human and murine TLR3 extracellulardomains have been determined ((Bell et al., Proc. Natl. Acad. Sci.(USA), 102:10976-80, 2005; Choe, et al., Science 309:581-585, 2005; Liuet al., Science, 320:379-381, 2008). TLR3 adopts the overall shape of asolenoid horseshoe decorated by glycans and has 23 tandem units ofleucine-rich repeat (LRR) motifs. The dsRNA binding sites have beenmapped to two distinct regions (Liu et al., Science, 320:379-81, 2008).The singaling assembly has been proposed to consist of 1 dsRNA and twoTLR3 extracellular domains (Leonard et al., Proc. Natl. Acad. Sci. (USA)105: 258-263, 2008).

TLR3 has been shown to drive pathogenic mechanisms in a spectrum ofinflammatory, immune-mediated and autoimmune diseases including, forexample, septic shock (Cavassani et al., J. Exp. Med. 205:2609-2621,2008), acute lung injury (Murray et al., Am. J. Respir. Crit. Care Med.178:1227-1237, 2008), rheumatoid arthritis (Kim et al., Immunol. Lett.124:9-17, 2009; Brentano et al., Arth. Rheum. 52:2656-2665, 2005),asthma (Sugiura et al., Am. J. Resp. Cell Mol. Biol. 40:654-662, 2009;Morishima et al., Int. Arch. Allergy Immunol. 145:163-174, 2008; Stowellet al., Respir. Res. 10:43, 2009), inflammatory bowel disease such asCrohn's disease and ulcerative colitis (Zhou et al., J. Immunol.178:4548-4556, 2007; Zhou et al., Proc. Natl. Acad. Sci. (USA)104:7512-7515, 2007), autoimmune liver disease (Lang et al., J. Clin.Invest. 116:2456-2463, 2006) and type I diabetes (Dogusan et al.Diabetes 57:1236-1245, 2008; Lien and Zipris, Curr. Mol. Med. 9:52-68,2009). Furthermore, organ-specific increases in TLR3 expression havebeen shown to correlate with a number of pathological conditions drivenby dysregulated local inflammatory responses such as in liver tissue inprimary biliary cirrhosis (Takii et al., Lab Invest. 85:908-920, 2005),rheumatoid arthritis joints (Ospelt et al., Arthritis Rheum.58:3684-3692, 2008), and nasal mucosa of allergic rhinitis patients(Fransson et al., Respir. Res. 6:100, 2005).

In necrotic conditions, the release of intracellular content includingendogenous mRNA triggers secretion of cytokines, chemokines and otherfactors that induce local inflammation, facilitate clearance of deadcell remnants and repair the damage. Necrosis often perpetuatesinflammatory processes, contributing to chronic or exaggeratedinflammation (Bergsbaken et al., Nature Reviews 7:99-109, 2009).Activation of TLR3 at the site of necrosis may contribute to theseaberrant inflammatory processes and generate a further pro-inflammatorypositive feedback loop via the released TLR3 ligands. Thus, TLR3antagonism may be beneficial in a variety of disorders involving chronicor exaggerated inflammation and/or necrosis.

Down-modulation of TLR3 activation may also represent a novel treatmentstrategy for oncologic indications including renal cell carcinomas andhead and neck squamous cell carcinomas (Morikawa et al., Clin. CancerRes. 13:5703-5709, 2007; Pries et al., Int. J. Mol. Med. 21:209-215,2008). Furthermore, the TLR3^(L423F) allele encoding a protein withreduced activity has been associated with protection against advanced“dry” age-related macular degeneration (Yang et al., N. Engl. J. Med.359:1456-1463, 2008), indicating that TLR3 antagonists may be beneficialin this disease.

Pathologies associated with inflammatory conditions and others, such asthose associated with infections, have significant health and economicimpacts. Yet, despite advances in many areas of medicine, comparativelyfew treatment options and therapies are available for many of theseconditions.

Thus, a need exists to suppress TLR3 activity to treat TLR3-associatedconditions.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1 shows the effect of anti-human TLR3 (huTLR3) mAbs in an NF-κBreporter gene assay.

FIGS. 2A and 2B show the effect (% inhibition) or anti-huTLR3 mAbs in aBEAS-2B assay.

FIGS. 3A and 3B show the effect of anti-huTLR3 mAbs in a NHBE assay.

FIG. 4 shows the effect of anti-huTLR3 mAbs in a PBMC assay.

FIGS. 5A and 5B show the effect of anti-huTLR3 mAbs in a HASM assay.

FIGS. 6A, 6B and 6C show the binding of anti-huTLR3 mAbs to TLR3mutants.

FIG. 7A shows epitopes for mAb 15EVQ (black) and C1068 mAb (grey) (topimage) and epitope for mAb 12QVQ/QSV (black, bottom image) superimposedon the structure of human TLR3 ECD. FIG. 7B shows localized H/D exchangeperturbation map of TLR3 ECD protein complexed with mAb 15EVQ.

FIGS. 8A and 8B show the effect of rat/mouse anti-mouse TLR3 mAb mAb5429 (surrogate) in A) NF-κB and B) ISRE reporter gene assays.

FIG. 9 shows the effect of the surrogate mAbs (mAb 5429, mAb c1811) inthe MEF CXCL10/IP-10 assay.

FIG. 10 shows specificity of binding of the surrogate mAb to TLR3. Toppanel: isotype control; bottom panel: mAb c1811.

FIG. 11 shows effect of the surrogate mAbs on penH level in an AHRmodel.

FIG. 12 shows effect of the surrogate mAbs on total neutrophil numbersin BAL fluid in an AHR model.

FIG. 13 shows effect of the surrogate mAbs on CXCL10/IP-10 levels in BALfluid in an AHR model.

FIG. 14 shows effect of the surrogate mAb on histopathology scores in aDSS model.

FIGS. 15A and 15B show effect of the surrogate mAb on 15A histopathologyscores and 15B neutrophil influx in a T-cell transfer model.

FIG. 16 shows effect of the surrogate mAb on clinical scores in a CIAmodel.

FIG. 17 shows effect of the surrogate mAb on the clinical AUC scores ina CIA model.

FIG. 18 shows effect of the surrogate mAb on the survival of C57BL/6mice following intranasal administration of influenza A/PR/8/34. mAbdosing began at day −1.

FIG. 19 shows effect of the surrogate mAb on clinical scores followinginfluenza A/PR/8/34 administration. mAb dosing began at day −1.

FIG. 20 shows effect of the surrogate mAb on body weight over 14 daysafter administration of influenza A/PR/8/34. mAb dosing began at day −1.

FIGS. 21A and 21B show effect of the surrogate mAbs on blood glucoselevels in 21A WT DIO and 21B TLR3KO DIO animals after glucose challenge.

FIG. 22 shows effect of the surrogate mAb on insulin levels in WT DIOanimals.

FIGS. 23A and 23B show effect of mAb 15EVQ on 23A NTHi and 23Brhinovirus induced CXCL10/IP-10 and CCL5/RANTES levels in NHBE cells.

FIGS. 24A and 24B show effect of mAb 15EVQ on 24A) sICAM-1 levels and24B viability in HUVEC cells.

FIG. 25 shows survival of animals following administration of thesurrogate mAb 3 days post infection with influenza A.

FIG. 26 shows clinical scores following administration of the surrogatemAb 3 days post infection with influenza A.

FIG. 27 shows body weight change of animals following administration ofthe surrogate mAb 3 days post infection with influenza A.

FIGS. 28A and 28B show the molecular structure of the quaternary complexof huTLR3 ECD with Fab 12QVQ/QSV, Fab 15EVQ and Fab c1068 in 28A. inribbon and surface representations. The TLR3 ECD is in light gray withthe N-terminus labeled N; all Fab molecules are shown in dark gray inribbons representation. 28B. The epitopes are colored light gray andlabeled on the TLR3 ECD as for the Fabs in 28A. In FIGS. 28A and 28B,29A and 29B and 30A, 30B and 30C, the Fab 12QVQ/QSV, Fab c1068 and Fab15EVQ are abbreviated to Fab12, Fab1068 and Fab15, respectively in thelabels for clarity.

FIGS. 29A and 29B. Show a mechanism of neutralization by Fab 15EVQ. 29A.dsRNA:TLR3 signaling unit (SU) is shown with the Fab 15EVQ epitopehighlighted (light gray) in one of the two TLR3 ECD (light and darkgray, and labeled TLR3). The dsRNA ligand is shown as a double helix inlight gray. 29B. An illustration of Fab 15EVQ binding that stericallyinhibited dsRNA binding and thus, inhibits the formation of the SU.Binding of Fab 15EVQ, which is higher affinity, will prevent the SU fromforming or will disassemble the pre-formed SU.

FIGS. 30A, 30B and 30C show a mechanism of Fab 12QVQ/QSV and Fab c1068and clustering of TLR3 signaling units (SU). 30A. Fab 12QVQ/QSV and Fabc1068 can bind (or co-bind) a single SU. 30B. Model for closestclustering of two SUs on a dsRNA of about 76 base pairs. The threeepitopes are highlighted in different molecules for clarity. 30C.Binding of Fab 12QVQ/QSV and Fab c1068 prevents SU clustering due tosteric clashes between the antibodies and neighboring SUs. The twoleft-pointing arrows qualitatively represent different degrees ofseparation of SUs due to the antibodies (bottom arrow for Fab 12QVQ/QSVand top arrow for Fab c1068).

FIGS. 31A, 31B, 31C and 31D show the correspondence between sequential,Kabat, and Chothia numbering for exemplary antibodies. The CDRs and HVsare highlighted in gray.

FIG. 32 shows alignment of VL of mAb 15EVQ with human Vk1 frameworks.Chothia hypervariable loops are underlined, paratope residues doubleunderlined and the framework differences highlighted in gray. The Vκ1genes are *01 alleles unless otherwise indicated. Residue numbering issequential.

FIG. 33 shows alignment of VH of mAb 15EVQ with human Vh5 frameworks.Sequence features indicated as in FIG. 32.

FIGS. 34A and 34B show alignment of VL of mAb 12QVQ/QSV with human Vk3frameworks. Sequence features indicated as in FIG. 32.

FIG. 35 shows alignment of VL and VH of mAb 15EVQ or mAb 12QVQ/QSV withhuman Jκ, Jλ or Jλ frameworks. Sequence features indicated as in FIG.32.

SUMMARY OF THE INVENTION

One aspect of the invention is an isolated antibody or fragment thereof,wherein the antibody binds toll-like receptor 3 (TLR3) amino acidresidues K416, K418, L440, N441, E442, Y465, N466, K467, Y468, R488,R489, A491, K493, N515, N516, N517, H539, N541, 5571, L595, and K619 ofSEQ ID NO: 2.

Another aspect of the invention is an isolated antibody or fragmentthereof, wherein the antibody binds toll-like receptor 3 (TLR3) aminoacid residues 5115, D116, K117, A120, K139, N140, N141, V144, K145,T166, Q167, V168, S188, E189, D192, A195, and A219 of SEQ ID NO: 2.

Another aspect of the invention is an isolated antibody having a heavychain variable region and a light chain variable region or fragmentthereof, wherein the antibody binds TLR3 having an amino acid sequenceshown in SEQ ID NO: 2 with the heavy chain variable region Chothiaresidues W33, F50, D52, D54, Y56, N58, P61, E95, Y97, Y100, and D100band the light chain variable region Chothia residues Q27, Y32, N92, T93,L94, and S95.

Another aspect of the invention is an isolated antibody having a heavychain variable region and a light chain variable region or fragmentthereof, wherein the antibody binds TLR3 having an amino acid sequenceshown in SEQ ID NO: 2 with the heavy chain variable region Chothiaresidues N31a, Q52, R52b, S53, K54, Y56, Y97, P98, F99, and Y100, andthe light chain variable region Chothia residues G29, S30, Y31, Y32,E50, D51, Y91, D92, and D93.

Another aspect of the invention is an isolated antibody reactive withTLR3, wherein the antibody has at least one of the following properties:

-   -   a. binds to human TLR3 with a Kd fo<10 nM;    -   b. reduces human TLR3 biological activity in an in vitro        poly(I:C) NF-kB reporter gene assay >50% at 1 μg/ml;    -   c. inhibits >60% of IL-6 or CXCL10/IP-10 production from BEAS-2B        cells stimulated with <100 ng/ml poly(I:C) at 10 μg/ml;    -   d. inhibits >50% of IL-6 or CXCL10/IP-10 production from BEAS-2B        cells stimulated with <100 ng/ml poly(I:C) at 0.4 μg/ml;    -   e. inhibits >50% of IL-6 production from NHBE cells stimulated        with 62.5 ng/ml poly(I:C) at 5 μg/ml;    -   f. inhibits >50% of IL-6 production from NHBE cells stimulated        with 62.5 ng/ml poly(I:C) at 1 μg/ml;    -   g. inhibits >20% of poly(I:C)-induced IFN-γ, IL-6 or IL-12        production by PBMC cells at 1 μg/ml;    -   h. inhibits cynomologus TLR3 biological activity in an in vitro        NF-kB reporter gene assay with IC50<10 μg/ml; or    -   i. inhibits cynomologus TLR3 biological activity in an in vitro        ISRE reporter gene assay with IC50<5 μg/ml.

Another aspect of the invention is an isolated antibody reactive withTLR3 that competes for TLR3 binding with a monoclonal antibody, whereinthe monoclonal antibody comprises the amino acid sequences of certainheavy chain complementarity determining regions (CDRs) 1, 2 and 3, theamino acid sequences of certain light chain CDRs 1, 2 and 3, the aminoacid sequences of certain heavy chain variable regions (VH) or the aminoacid sequence of certain light chain variable regions (VL).

Another aspect of the invention is an isolated antibody reactive withTLR3 comprising both a heavy chain variable region and a light chainvariable region and wherein the antibody comprises the amino acidsequences of certain heavy chain complementarity determining regions(CDRs) 1, 2 and 3 and the amino acid sequences of certain light chainCDRs 1, 2 and 3.

Another aspect of the invention is an isolated antibody reactive withTLR3 comprising both a heavy chain variable region and a light chainvariable region and wherein the antibody comprises the amino acidsequences of certain heavy chain variable regions (VH) and the aminoacid sequences of certain light chain variable regions (VL).

Another aspect of the invention is an isolated antibody reactive withTLR3 comprising both a heavy chain variable region and a light chainvariable region and wherein the antibody comprises the amino acidsequence of certain heavy chains and the amino acid sequence of certainlight chains.

Another aspect of the invention is an isolated antibody heavy chaincomprising the amino acid sequence shown in SEQ ID NO: 6, 8, 10, 12, 14,16, 18, 20, 22, 24, 26, 28, 30, 32, 34, 36, 38, 40, 42, 124, 125, 126,127, 128, 129, 159, 198, 200, 202, 164, 212, 213, 214, 215 or 216.

Another aspect of the invention is an isolated antibody light chaincomprising the amino acid sequence shown in SEQ ID NO: 5, 7, 9, 11, 13,15, 17, 19, 21, 23, 25, 27, 29, 31, 33, 35, 37, 39, 41, 122, 123, 197,199, 201, 163, 209, 210, 211, or 225. Another aspect of the invention isan isolated antibody heavy chain comprising the amino acid sequenceshown in SEQ ID NO: 102, 130, 131, 132, 133, 134, 135, 160, 204, 206,208, 220, 166 or 168.

Another aspect of the invention is an isolated antibody light chaincomprising the amino acid sequence shown in SEQ ID NO: 155, 156, 157,158, 203, 205, 207, 165, 167, or 227.

Another aspect of the invention is an isolated polynucleotide encodingan antibody heavy chain comprising the amino acid sequence shown in SEQID NO: 6, 8, 10, 12, 14, 16, 18, 20, 22, 24, 26, 28, 30, 32, 34, 36, 38,40, 42, 124, 125, 126, 127, 128, 129, 159, 198, 200, 202, 164, 212, 213,214, 215 or 216.

Another aspect of the invention is an isolated polynucleotide encodingan antibody light chain comprising the amino acid sequence shown in SEQID NO: 5, 7, 9, 11, 13, 15, 17, 19, 21, 23, 25, 27, 29, 31, 33, 35, 37,39, 41, 122, 123, 197, 199, 201, 163, 209, 210, 211, or 225.

Another aspect of the invention is an isolated polynucleotide encodingan antibody heavy chain comprising the amino acid sequence shown in SEQID NO: 102, 130, 131, 132, 133, 134, 135, 160, 204, 206, 208, 220, 166or 168.

Another aspect of the invention is an isolated polynucleotide encodingan antibody light chain comprising the amino acid sequence shown in SEQID NO: 155, 156, 157, 158, 203, 205, 207, 165, 167, or 227.

Another aspect of the invention is a pharmaceutical compositioncomprising the isolated antibody of the invention and a pharmaceuticallyacceptable carrier.

Another aspect of the invention is a vector comprising at least onepolynucleotide of the invention.

Another aspect of the invention is a host cell comprising the vector ofthe invention.

Another aspect of the invention is a method of making an antibodyreactive with TLR3 comprising culturing the host cell of the inventionand recovering the antibody produced by the host cell.

Another aspect of the invention is a method of treating or preventing aninflammatory condition comprising administering a therapeuticallyeffective amount of the isolated antibody of the invention to a patientin need thereof for a time sufficient to treat or prevent theinflammatory condition.

Another aspect of the invention is a method of treating or preventing asystemic inflammatory condition comprising administering atherapeutically effective amount of the isolated antibody of theinvention to a patient in need thereof for a time sufficient to treat orprevent the systemic inflammatory condition.

Another aspect of the invention is a method of treating type II diabetescomprising administering a therapeutically effective amount of theisolated antibody of the invention to a patient in need thereof for atime sufficient to treat type II diabetes.

Another aspect of the invention is a method of treating hyperglycemiacomprising administering a therapeutically effective amount of theisolated antibody of the invention to a patient in need thereof for atime sufficient to treat the hyperglycemia.

Another aspect of the invention is a method of treating hyperinsulinemiacomprising administering a therapeutically effective amount of theisolated antibody of the invention to a patient in need thereof for atime sufficient to treat the insulin resistance.

Another aspect of the invention is a method of treating or preventingviral infections comprising administering a therapeutically effectiveamount of the isolated antibody of the invention to a patient in needthereof for a time sufficient to treat or prevent viral infections.

DETAILED DESCRIPTION OF THE INVENTION

All publications, including but not limited to patents and patentapplications, cited in this specification are herein incorporated byreference as though fully set forth.

The term “antagonist” as used herein means a molecule that partially orcompletely inhibits, by any mechanism, an effect of another moleculesuch as a receptor or intracellular mediator.

As used herein, a “TRL3 antibody antagonist” or an antibody “reactivewith TLR3” describes an antibody that is capable of, directly orindirectly, substantially counteracting, reducing or inhibiting TLR3biological activity or TLR3 receptor activation. For example, anantibody reactive with TLR3 can bind directly to TLR3 and neutralizeTLR3 activity, i.e, block TLR3 signaling to reduce cytokine andchemokine release or NF-κB activation.

The term “antibodies” as used herein is meant in a broad sense andincludes immunoglobulin or antibody molecules including polyclonalantibodies, monoclonal antibodies including murine, human,human-adapted, humanized and chimeric monoclonal antibodies and antibodyfragments.

In general, antibodies are proteins or peptide chains that exhibitbinding specificity to a specific antigen. Intact antibodies areheterotetrameric glycoproteins, composed of two identical light chainsand two identical heavy chains. Typically, each light chain is linked toa heavy chain by one covalent disulfide bond, while the number ofdisulfide linkages varies between the heavy chains of differentimmunoglobulin isotypes. Each heavy and light chain also has regularlyspaced intrachain disulfide bridges. Each heavy chain has at one end avariable domain (variable region) (VH) followed by a number of constantdomains (constant regions). Each light chain has a variable domain atone end (VL) and a constant domain at its other end; the constant domainof the light chain is aligned with the first constant domain of theheavy chain and the light chain variable domain is aligned with thevariable domain of the heavy chain. Antibody light chains of anyvertebrate species can be assigned to one of two clearly distinct types,namely kappa (κ) and lambda (λ), based on the amino acid sequences oftheir constant domains.

Immunoglobulins can be assigned to five major classes, namely IgA, IgD,IgE, IgG and IgM, depending on the heavy chain constant domain aminoacid sequence. IgA and IgG are further sub-classified as the isotypesIgA₁, IgA₂, IgG₁, IgG₂, IgG₃ and IgG₄.

The term “antibody fragments” means a portion of an intact antibody,generally the antigen binding or variable region of the intact antibody.Examples of antibody fragments include Fab, Fab′, F(ab′)₂ and Fvfragments, diabodies, single chain antibody molecules and multispecificantibodies formed from at least two intact antibodies.

An immunoglobulin light chain variable region or heavy chain variableregion consists of a “framework” region interrupted by three“antigen-binding sites”. The antigen-binding sites are defined usingvarious terms as follows: (i) the term Complementarity DeterminingRegions (CDRs) is based on sequence variability (Wu and Kabat, J. Exp.Med. 132:211-250, 1970). Generally, the antigen-binding site has sixCDRs; three in the VH (HCDR1, HCDR2, HCDR3), and three in the VL (LCDR1,LCDR2, LCDR3) (Kabat et al., Sequences of Proteins of ImmunologicalInterest, 5th Ed. Public Health Service, National Institutes of Health,Bethesda, Md., 1991). (ii) The term “hypervariable region”, “HVR”, or“HV” refers to the regions of an antibody variable domain which arehypervariable in structure as defined by Chothia and Lesk (Chothia andLesk, Mol. Biol. 196:901-917, 1987). Generally, the antigen-binding sitehas six hypervariable regions, three in VH (H1, H2, H3) and three in VL(L1, L2, L3). Chothia and Lesk refer to structurally conserved HVs as“canonical structures”. (iii) The “IMGT-CDRs” as proposed by Lefranc(Lefranc et al., Dev. Comparat. Immunol. 27:55-77, 2003) are based onthe comparison of V domains from immunoglobulins and T-cell receptors.The International ImMunoGeneTics (IMGT) database (http://www_imgt_org)provides a standardized numbering and definition of these regions. Thecorrespondence between CDRs, HVs and IMGT delineations is described inLefranc et al., Dev. Comparat. Immunol. 27:55-77, 2003. (iv) Theantigen-binding site can also be delineated based on SpecificityDetermining Residue Usage (SDRU)(Almagro, Mol. Recognit. 17:132-143,2004), where Specificity Determining Residues (SDR), refers to aminoacid residues of an immunoglobulin that are directly involved in antigencontact. SDRU is a precise measure of a number and distribution of SDRfor different types of antigens as defined by analyses of crystalstructures of antigen-antibody complexes. (v) The antigen-binding sitecan also be defined as the antibody paratope residues identified fromcrystal structure of the antigen-antibody complex.

The term “composite sequences” as used herein means an antigen-bindingsite defined to include all amino acid residues delineated individuallyby Kabat, Chothia or IMGT, or any other suitable antigen-binding sitedelineation.

“Chothia residues” as used herein are the antibody VL and VH residuesnumbered according to Al-Lazikani (Al-Lazikani et al., J. Mol. Biol.273:927-48, 1997). Correspondence between the two most used numberingsystems, Kabat (Kabat et al., Sequences of Immunological Interest,5^(th) Ed. Public Health Service, NIH, Bethesda, Md., 1991) and Chothia(Chothia and Lesk, Mol. Biol. 196:901-17, 1987) in relation tosequential polypeptide numbering is shown in FIGS. 31A-31D for exemplaryantibodies of the invention.

“Framework” or “framework sequences” are the remaining sequences of avariable region other than those defined to be antigen-binding site. Theframework is typically divided into four regions, FR1, FR2, FR3, andFR3, which form a scaffold for the three antigen-binding sites in eachvariable region. Because the antigen-binding site can be defined byvarious terms as described above, the exact amino acid sequence of aframework depends on how the antigen-binding site was defined.

“A light chain variable region kappa 1 (Vκ1) framework” or “Vκ1” as usedherein refers to a framework having an amino acid sequence encoded byany of the human Vκ1 functional genes or alleles thereof. Exemplaryfunctional human Vκ1 genes are IGKV1-5*01, IGKV1-6*01, IGKV1-8*01,IGKV1-9*01, IGKV1-12*01, IGKV1-13*02, IGKV1-16*01, IGKV1-17*01,IGKV1-27*01, IGKV1-33*01, IGKV1-37*01, IGKV1-39*01, IGKV1D-8*01,IGKV1D-12*01, IGKV1D-13*01, IGKV1D-16*01, IGKV1D-17*01, IGKV1D-33*01,IGKV1D-37*01, IGKV1D-39*01, IGKV1D-42*01, or IGKV1D-43*01. Nomenclatureof the immunoglobulin genes is well known.

“A light chain variable region lambda 3 (Vλ3) framework” or “Vλ3” asused herein refers to a framework having an amino acid sequence encodedby any of the human Vλ3 functional genes or alleles thereof. Exemplaryfunctional human Vλ3 genes are IGLV3-1*01, IGLV3-9*01, IGLV3-10*01,IGLV3-12*01, IGLV3-16*01, IGLV3-19*01, IGLV3-21*01, IGLV3-22*01,IGLV3-25*01, IGLV3-27*01, and IGLV3-32*01.

“A heavy chain variable region Vh5 framework” or “Vh5” as used hereinrefers to a framework having an amino acid sequence encoded by any ofthe human Vh5 functional genes or alleles thereof. Exemplary functionalhuman Vh5 genes are IGHV5-51*01 and IGHV5-1*01.

“A heavy chain variable region Vh6 framework” or “Vh6” as used hereinrefers to a framework having an amino acid sequence encoded by any ofthe human Vh6 functional genes or alleles thereof. An exemplaryfunctional human Vh6 gene is IGHV6-1*01.

“A light chain kappa J-region (Jκ) framework” or “Jκ” as used hereinrefers to a framework having an amino acid sequence encoded by any ofthe human Jκ functional genes or alleles thereof. Exemplary functionalhuman Vκ genes are IGKJ1, IGKJ2, IGKJ3, IGKJ4, and IGKJ5.

“A light chain lambda J-region (Jλ) framework” or “Jλ” as used hereinrefers to a framework having an amino acid sequence encoded by any ofthe human Jλ functional genes or alleles thereof. Exemplary functionalhuman Jλ genes are IGLJ1, IGLJ2, IGLJ3, IGLJ4, IGLJ5, IGLJ6, and IGLJ7.

“A heavy chain J-region (Jh) framework” or “Jh” as used herein refers toa framework having an amino acid sequence encoded by any of the human Jhfunctional genes or alleles thereof. Exemplary functional human Jh genesare IGHJ1, IGHJ2, IGHJ3, IGHJ4, IGHJ5, and IGHJ6.

“Germline genes” or “antibody germline genes” as used herein areimmunoglobulin sequences encoded by non-lymphoid cells that have notundergone the maturation process that leads to genetic rearrangement andmutation for expression of a particular immunoglobulin.

“Scaffold” as used herein refers to amino acid sequences of light orheavy chain variable regions encoded by human germline genes. Thus, thescaffold encompasses both the framework and the antigen-binding site.

The term “antigen” as used herein means any molecule that has theability to generate antibodies either directly or indirectly. Includedwithin the definition of “antigen” is a protein-encoding nucleic acid.

The term “homolog” means protein sequences having between 40% and 100%sequence identity to a reference sequence. Homologs of human TLR3include polypeptides from other species that have between 40% and 100%sequence identity to a known human TLR3 sequence. Percent identitybetween two peptide chains can be determined by pairwise alignment usingthe default settings of the AlignX module of Vector NTI v.9.0.0(Invitrogen, Carlsbad, Calif.). By “TLR3” is meant human TLR3 (huTLR3)and its homologs. The nucleotide and amino acid sequences of the fulllength huTLR3 are shown in SEQ ID NOs: 1 and 2, respectively. Thenucleotide and amino acid sequences of the huTLR3 extracellular domain(ECD) are shown in SEQ ID NOs: 3 and 4, respectively.

The term “substantially identical” as used herein means that the twoantibody or antibody fragment amino acid sequences being compared areidentical or have “insubstantial differences”. Insubstantial differencesare substitutions of 1, 2, 3, 4, 5 or 6 amino acids in an antibody orantibody fragment amino acid sequence. Amino acid sequencessubstantially identical to the sequences disclosed herein are also partof this application. In some embodiments, the sequence identity can beabout 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or higher.Percent identity can be determined as described above. Exemplary peptidechains being compared are heavy or light chain variable regions.

The term “in combination with” as used herein means that the describedagents can be administered to an animal together in a mixture,concurrently as single agents or sequentially as single agents in anyorder.

The term “inflammatory condition” as used herein means a localizedresponse to cellular injury that is mediated in part by the activity ofcytokines, chemokines, or inflammatory cells (e.g., neutrophils,monocytes, lymphocytes, macrophages) which is characterized in mostinstances by pain, redness, swelling, and loss of tissue function. Theterm “inflammatory pulmonary condition” as used herein means aninflammatory condition affecting or associated with the lungs.

The term “monoclonal antibody” (mAb) as used herein means an antibody(or antibody fragment) obtained from a population of substantiallyhomogeneous antibodies. Monoclonal antibodies are highly specific,typically being directed against a single antigenic determinant. Themodifier “monoclonal” indicates the substantially homogeneous characterof the antibody and does not require production of the antibody by anyparticular method. For example, murine mAbs can be made by the hybridomamethod of Kohler et al., Nature 256:495-497, 1975. Chimeric mAbscontaining a light chain and heavy chain variable region derived from adonor antibody (typically murine) in association with light and heavychain constant regions derived from an acceptor antibody (typicallyanother mammalian species such as human) can be prepared by the methoddisclosed in U.S. Pat. No. 4,816,567. Human-adapted mAbs having CDRsderived from a non-human donor immunoglobulin (typically murine) and theremaining immunoglobulin-derived parts of the molecule being derivedfrom one or more human immunoglobulins can be prepared by techniquesknown to those skilled in the art such as that disclosed in U.S. Pat.No. 5,225,539. Human framework sequences useful for human-adaptation canbe selected from relevant databases by those skilled in the art.Optionally, human-adapted mAbs can be further modified by incorporatingaltered framework support residues to preserve binding affinity bytechniques such as those disclosed in Queen et al., Proc. Natl. Acad.Sci. (USA), 86:10029-10032, 1989 and Hodgson et al., Bio/Technology,9:421, 1991.

Fully human mAbs lacking any non-human sequences can be prepared fromhuman immunoglobulin transgenic mice by techniques referenced in, e.g.,Lonberg et al., Nature 368:856-859, 1994; Fishwild et al., NatureBiotechnology 14:845-851, 1996; and Mendez et al., Nature Genetics15:146-156, 1997. Human mAbs can also be prepared and optimized fromphage display libraries by techniques referenced in, e.g., Knappik etal., J. Mol. Biol. 296:57-86, 2000; and Krebs et al., J. Immunol. Meth.254:67-84 2001. Fragments of antibodies e.g., Fab, F(ab′)2, Fd, and dAbfragments may be produced by cleavage of the antibodies or byrecombinant engineering. For example, Fab and F(ab′)2 fragments may begenerated by treating the antibodies with an enzyme such as pepsin.

The term “epitope” as used herein means a portion of an antigen to whichan antibody specifically binds. Epitopes usually consist of chemicallyactive (such as polar, non-polar or hydrophobic) surface groupings ofmoieties such as amino acids or polysaccharide side chains and can havespecific three-dimensional structural characteristics, as well asspecific charge characteristics. An epitope can be linear in nature orcan be a discontinous epitope, e.g., a conformational epitope, which isformed by a spatial relationship between non-contiguous amino acids ofan antigen rather than a linear series of amino acids. A conformationalepitope includes epitopes resulting from folding of an antigen, whereamino acids from differing portions of the linear sequence of theantigen come in close proximity in 3-dimensional space.

The term “paratope” as used herein refers to a portion of an antibody towhich an antigen specifically binds. A paratope can be linear in natureor can be discontinuous, formed by a spatial relationship betweennon-contiguous amino acids of an antibody rather than a linear series ofamino acids. A “light chain paratope” and a “heavy chain paratope” or“light chain paratope amino acid residues” and “heavy chain paratopeamino acid residues” refer to antibody light chain and heavy chainresidues in contact with an antigen, respectively.

The term “specific binding” as used herein refers to antibody binding toa predetermined antigen with greater affinity than for other antigens orproteins. Typically, the antibody binds with a dissociation constant(K_(D)) of 10⁻⁷ M or less, and binds to the predetermined antigen with aK_(D) that is at least twofold less than its K_(D) for binding to anon-specific antigen (e.g., BSA, casein, or any other specifiedpolypeptide) other than the predetermined antigen. The phrases “anantibody recognizing an antigen” and “an antibody specific for anantigen” are used interchangeably herein with the term an antibody whichbinds specifically to an antigen” or “an antigen specific antibody” e.g.a TLR3 specific antibody. The dissociation constant can be measuredusing standard procedures as described below.

The term “TLR3 biological activity” or “TLR3 activation” as used hereinrefers to any activity occurring as a result of ligand binding to TLR3.TLR3 ligands include dsRNA, poly(I:C), and endogenous mRNA, e.g.,engodenous mRNA released from necrotic cells. An exemplary TLR3activation results in activation of NF-κB in response to the TLR3ligand. NF-κB activation can be assayed using a reporter-gene assay uponinduction of the receptor with poly(I:C) (Alexopoulou et al., Nature413:732-738, 2001; Hacker et al., EMBO J. 18:6973-6982, 1999). Anotherexemplary TLR3 activation results in activation of interferon responsefactors (IRF-3, IRF-7) in response to the TLR3 ligand. TLR3-mediated IRFactivation can be assayed using a reporter gene driven by aninterferon-stimulated response element (ISRE). Another exemplary TLR3activation results in secretion of pro-inflammatory cytokines andchemokines, for example TNF-α, IL-6, IL-8, IL-12, CXCL5/IP-10 andRANTES. The release of cytokines and chemokines from cells, tissues orin circulation can be measured using well-known immunoassays, such as anELISA immunoassay.

Conventional one and three-letter amino acid codes are used herein asfollows:

Amino acid Three-letter code One-letter code Alanine ala A Arginine argR Asparagine asn N Aspartate asp D Cysteine cys C Glutamate glu EGlutamine gln Q Glycine gly G Histidine his H Isoleucine ile I Leucineleu L Lysine lys K Methionine met M Phenylalanine phe F Proline pro PSerine ser S Threonine thr T Tryptophan trp W Tyrosine tyr Y Valine valV

Compositions of Matter

The present invention provides antibody antagonists capable ofinhibiting TLR3 biological activity and uses of such antibodies. SuchTLR3 antagonists may have the properties of binding TLR3 and inhibitingTLR3 activation. Exemplary mechanisms by which TLR3 activation may beinhibited by such antibodies include in vitro, in vivo or in situinhibition of ligand binding to TLR3, inhibition of receptordimerization, inhibition of TLR3 localization to the endosomalcompartment, inhibition of kinase activity of downstream signalingpathways, or inhibition of TLR3 mRNA transcription. Other antibodyantagonists capable of inhibiting TLR3 activation by other mechanismsare also within the scope of the various aspects and embodiments of theinvention. These antagonists are useful as research reagents, diagnosticreagents and therapeutic agents.

Antibody diversity, in a natural system, is created by the use ofmultiple germline genes encoding variable regions and a variety ofsomatic events. The somatic events include recombination of variablegene segments with diversity (D) and joining (J) gene segments to make acomplete VH region, and the recombination of variable and joining genesegments to make a complete VL region. The recombination process itselfcan be imprecise, resulting in the loss or addition of amino acids atthe V(D)J junctions. These mechanisms of diversity occur in thedeveloping B cell prior to antigen exposure. After antigenicstimulation, the expressed antibody genes in B cells undergo somaticmutation. Based on the estimated number of germline gene segments, therandom recombination of these segments, and random VH-VL pairing, up to1.6×10⁷ different antibodies could be produced (Fundamental Immunology,3rd ed. (1993), ed. Paul, Raven Press, New York, N.Y.). When otherprocesses that contribute to antibody diversity (such as somaticmutation) are taken into account, it is thought that upwards of 10¹⁰different antibodies could be generated (Immunoglobulin Genes, 2nd ed.(1995), eds. Jonio et al., Academic Press, San Diego, Calif.). Becauseof the many processes involved in generating antibody diversity, it ishighly unlikely that independently derived monoclonal antibodies withthe same antigen specificity will have identical amino acid sequences.

The invention provides novel antigen-binding sites and immunoglobulinchains derived from human immunoglobulin gene libraries. The structurefor carrying an antigen-binding site is generally an antibody heavy orlight chain or portion thereof, where the antigen-binding site islocated to a naturally occurring antigen-binding site as determined asdescribed above.

The invention provides an isolated antibody or fragment thereof reactivewith TLR3 comprising both a heavy chain and a light chain variableregion and wherein the antibody comprises the heavy chaincomplementarity determining region (CDR) amino acid sequences 1, 2 and 3(HCDR1, HCDR2 and HCDR3) and the light chain complementarity determiningregion (CDR) amino acid sequences 1, 2 and 3 (LCDR1, LCDR2 and LCDR3) asshown in Table 1a.

TABLE 1a SEQ ID NO: mAb no: HCDR1 HCDR2 HCDR3 LCDR1 LCDR2 LCDR3 16 52 8854 49 50 51 17 58 64 60 55 56 57 18 70 77 72 67 68 69 19 82 83 84 79 8089  1 46 47 48 43 44 45  2 52 53 54 49 50 51  3 58 59 60 55 56 57  4 6162 60 55 56 57  5 61 64 60 55 56 63  6 61 64 60 55 56 65  7 61 64 60 5556 66  8 70 71 72 67 68 69  9 70 73 72 67 68 69 10 70 75 72 67 68 74 1170 77 72 67 68 76 12 70 77 72 67 68 78 13 82 83 84 79 80 81 14 82 86 8479 80 85 15* 82 86 84 79 80 87 15** 111 112 84 109 110 113 15-1 111 11484 109 110 113 15-2 115 112 84 109 110 113 15-3 116 112 84 109 110 11315-4 111 117 84 109 110 113 15-5 116 118 84 109 110 113 15-6 116 112 119109 110 113 15-7 111 112 84 120 110 113 15-8 111 112 84 121 110 113 15-9116 118 119 109 110 113 15-10 116 112 119 79 80 226 F17 61 192 60 55 56191 F18 70 194 72 67 68 193 F19 82 196 84 79 80 195 15* CDRs defined byIMGT 15** CDRs defined as consensus

In certain embodiments the invention provides an isolated antibody orfragment reactive with TLR3 comprising both a heavy chain variableregion and a light chain variable region and wherein the antibodycomprises a HCDR2 amino acid sequence as shown in SEQ ID NO: 192,wherein the HCDR2 of SEQ ID NO: 192 is defined as shown in Formula (I):

Xaa₆-I-Xaa₇-Xaa₈-R—S-Xaa₉-W—Y—N-D-Y-A-V—S—V—K—S,   (I)

-   -   wherein    -   Xaa₈ may be Arg or Lys;    -   Xaa₇ may be Tyr, His or Ser;    -   Xaa₈ may be Met, Arg or Tyr; and    -   Xaa₉ may be Lys or Arg.

In other embodiments, the invention provides an isolated antibody orfragment reactive with TLR3 comprising both a heavy chain variableregion and a light chain variable region and wherein the antibodycomprises a HCDR2 amino acid sequence as shown in SEQ ID NO: 194,wherein the HCDR2 of SEQ ID NO: 194 is defined as shown in Formula(III):

I-I-Q-Xaa₁₅-R—S—K—W—Y—N-Xaa₁₆-Y-A-Xaa₁₇-S—V—K—S,   (III)

-   -   wherein    -   Xaa₁₅ may be Lys, Thr or Ile;    -   Xaa₁₆ may be Asn or Asp; and    -   Xaa₁₇ may be Val or Leu.

In other embodiments, the invention provides an isolated antibody orfragment reactive with TLR3 comprising both a heavy chain variableregion and a light chain variable region and wherein the antibodycomprises a HCDR2 amino acid sequence as shown in SEQ ID NO: 196,wherein the HCDR2 of SEQ ID NO: 196 is defined as shown in Formula (V):

Xaa₂₄-I-D-P—S-D-S—Y-T-N—Y-Xaa₂₅-P—S—F-Q-G,   (V)

-   -   wherein    -   Xaa₂₄ may be Phe or Arg; and    -   Xaa₂₅ may be Ala or Ser.

In other embodiments, the invention provides an isolated antibody orfragment reactive with TLR3 comprising both a heavy chain variableregion and a light chain variable region and wherein the antibodycomprises a LCDR3 amino acid sequence as shown in SEQ ID NO: 191,wherein the LCDR3 of SEQ ID NO: 191 is defined as shown in Formula (II):

Xaa₁-S—Y-D-Xaa₂-Xaa₃-Xaa₄-Xaa₅-T-V,   (II)

-   -   wherein    -   Xaa₁ may be Ala, Gln, Gly or Ser;    -   Xaa₂ may be Gly, Glu or Ser;    -   Xaa₃ may be Asp or Asn;    -   Xaa₄ may be Glu or Ser; and    -   Xaa₅ may be Phe, Ala or Leu.

In other embodiments, the invention provides an isolated antibody orfragment reactive with TLR3 comprising both a heavy chain variableregion and a light chain variable region and wherein the antibodycomprises a LCDR3 amino acid sequence as shown in SEQ ID NO: 193,wherein the LCDR3 of SEQ ID NO: 193 is defined as shown in Formula (IV):

Xaa_(n)-S—Y-D-Xaa₁₁-P-Xaa₁₂-Xaa₁₃-Xaa₁₄-V,   (IV)

-   -   wherein    -   Xaa₁₃ may be Gln or Ser;    -   Xaa₁₁ may be Thr, Glu or Asp;    -   Xaa₁₂ may be Val or Asn;    -   Xaa₁₃ may be Tyr or Phe; and    -   Xaa₁₄ may be Ser, Asn or Gln.

In other embodiments, the invention provides an isolated antibody orfragment reactive with TLR3 comprising both a heavy chain variableregion and a light chain variable region and wherein the antibodycomprises a LCDR3 amino acid sequence as shown in SEQ ID NO: 195,wherein the LCDR3 of SEQ ID NO: 195 is defined as shown in Formula (VI):

Q-Q-Xaa₁₈-Xaa₁₉-Xaa₂₀-Xaa₂₁-Xaa₂₂-Xaa₂₃-T,   (VI)

-   -   wherein    -   Xaa₁₈ may be Tyr, Gly or Ala;    -   Xaa₁₉ may be Gly, Glu or Asn;    -   Xaa₂₀ may be Ser or Thr;    -   Xaa₂₁ may be Val, Ile or Leu;    -   Xaa₂₂ may be Ser or Leu; and    -   Xaa₂₃ may be Ile, Ser, Pro or Tyr.

The invention also provides an isolated antibody or fragment reactivewith TLR3 having the heavy chain complementarity determining region(CDR) amino acid sequences 1,2 and 3 (HCDR1, HCDR2 and HCDR3) and lightchain complementarity determining region (CDR) amino acid sequences 1, 2and 3 (LCDR1, LCDR2 and LCDR3) as shown in Table 1a.

Antibodies whose antigen-binding site amino acid sequences differinsubstantially from those shown in Table 1a (SEQ ID NOs: 49-121 and191-196) are encompassed within the scope of the invention. Typically,this involves one or more amino acid substitutions with an amino acidhaving similar charge, hydrophobic, or stereochemical characteristics.Additional substitutions in the framework regions, in contrast toantigen-binding sites may also be made as long as they do not adverselyaffect the properties of the antibody. Substitutions may be made toimprove antibody properties, for example stability or affinity. One,two, three, four, five or six substitutions can be made to the antigenbinding site. 1%, 2%, 3%, 4%, 5%, 10%, 15%, 20%, 25%, or 30% of theframework residues can be substituted, as long as the resulting antibodyretains desired properties.

Conservative modifications will produce molecules having functional andchemical characteristics similar to those of the molecule from whichsuch modifications are made. Substantial modifications in the functionaland/or chemical characteristics of the molecules may be accomplished byselecting substitutions in the amino acid sequence that differsignificantly in their effect on maintaining (1) the structure of themolecular backbone in the area of the substitution, for example, as asheet or helical conformation, (2) the charge or hydrophobicity of themolecule at the target site, or (3) the size of the molecule. Forexample, a “conservative amino acid substitution” may involve asubstitution of a native amino acid residue with a nonnative residuesuch that there is little or no effect on the polarity or charge of theamino acid residue at that position. Furthermore, any native residue inthe polypeptide may also be substituted with alanine, as has beenpreviously described for alanine scanning mutagenesis (MacLennan et al.,Acta Physiol. Scand. Suppl. 643:55-67, 1998; Sasaki et al., Adv.Biophys. 35:1-24, 1998). Desired amino acid substitutions (whetherconservative or non-conservative) can be determined by those skilled inthe art at the time such substitutions are desired. For example, aminoacid substitutions can be used to identify important residues of themolecule sequence, or to increase or decrease the affinity of themolecules described herein. Exemplary amino acid substitutions are shownin Table 1b.

In certain embodiments, conservative amino acid substitutions alsoencompass non-naturally occurring amino acid residues which aretypically incorporated by chemical peptide synthesis rather than bysynthesis in biological systems. Amino acid substitutions can be donefor example by PCR mutagenesis (U.S. Pat. No. 4,683,195). Libraries ofvariants can be generated using well known methods, for example usingrandom (NNK) or non-random codons, for example DVK codons, which encode11 amino acids (ACDEGKNRSYW), and screening the libararies for variantswith desired properties, as shown in Example 1. Table 1c showssubstitutions made to three parent TLR3 antibody antagonists within theLCDR3 and HCDR2 regions to improve antibody properties.

Depending on delineation of the antigen-binding sites, theantigen-binding site residues of the antibodies of the invention andsubsequently the framework residues may vary slightly for each heavy andlight chain.

TABLE 1b More Original Conservative residue Exemplary substitutionssubstitutions Ala (A) Val, Leu, Ile Val Arg (R) Lys, Gln, Asn Lys Asn(N) Gln Gln Asp (D) Glu Glu Cys (C) Ser, Ala Ser Gln (Q) Asn Asn Gly (G)Pro, Ala Ala His (H) Asn, Gln, Lys, Arg Arg Ile (I) Leu, Val, Met, Ala,Phe, Norleucine Leu Leu (L) Norleucine, Ile, Val, Met, Ala, Phe Ile Lys(K) Arg, 1,4 Diamino-butyric Acid, Gln, Asn Arg Met (M) Leu, Phe, IleLeu Phe (F) Leu, Val, Ile, Ala, Tyr Leu Pro (P) Ala Gly Ser (S) Thr,Ala, Cys Thr Thr (T) Ser Ser Trp (W) Tyr, Phe Tyr Tyr (Y) Trp, Phe, Thr,Ser Phe Val (V) Ile, Met, Leu, Phe, Ala, Norleucine Leu

Table 2a and 2b shows the antigen-binding site residues of exemplaryantibodies of the invention delineated according to Kabat, Chothia andIMGT, and their composite sequences.

In other embodiments, the invention provides an isolated antibody orfragment reactive with TLR3 comprising both a heavy chain variableregion and a light chain variable region and wherein the antibodycomprises the amino acid sequences of the heavy chain variable (VH) andthe light chain variable (VL) regions and also provides for eachisolated heavy chain variable and light chain variable region as shownin Table 3a. F17, F18 and F19 represent antibody variants comprisingconsensus amino acid sequences for families 17, 18 and 19, respectively(see Example 1).

TABLE 1C Family 17 SEQ ID mAb LCDR3 NO: 17 A S Y D G D E F T V 3 4 5 Q ES A 6 G S N S L 7 S S S L consensus A, Q, G, S S Y D G, E, S D, N E, SF, A, L T V 191 Family 17 SEQ ID mAb HCDR2 NO: 17 R I Y M R S K W Y N DY A V S V K S 3 H R 4 K S Y R 5 6 7 consensus R, K I Y, H, S M, R, Y R SK, R W Y N D Y A V S V K S 192 SEQ ID mAb LCDR3 NO: Family 18A 18 Q S YD S Q F S F G V 8 9 Family 18B 10 Q S Y D T P V Y S V 11 S E N F N 12 SD N F Q consensus Q, S S Y D T, E, D P V, N Y, F S, N, Q V 193 SEQFamily 18A, 18B ID mAb HCDR2 NO: 18 I I Q K R S K W Y N N Y A V S V K S8 T D 9 I D L 10 11 12 consensus I I Q K, T, I R S K W Y N N, D Y A V, LS V K S 194 Family 19 SEQ ID mAb LCDR2 NO: 19 Q Q Y G S V S I T 13 G E SI L S 14 A E T P 15 G N T L Y 15-1 G N T L Y 15-2 G N T L Y 15-3 G N T LY 15-4 G N T L Y 15-5 G N T L Y 15-6 G N T L Y 15-7 G N T L Y 15-8 G N TL Y 15-9 G N T L Y 15-10 G N T L Y consensus Q Q Y, G, A G, E, N S, T V,I, L S, L I, S, P, Y T 195 Family 19 SEQ ID mAb HCDR2 NO: 19 F I D P S DS Y T N Y A P S F Q G 13 14 15 15.1 R 15.2 15.3 15.4 S 15.5 R S 15.615.7 15-8 15-9 R S 15-10 consensus F, R I D P S D S Y T N Y A, S P S F QG 196 *consensus based on mAbs 10, 11, 12

Although the embodiments illustrated in the Examples comprise pairs ofvariable regions, one from a heavy and one from a light chain, a skilledartisan will recognize that alternative embodiments may comprise singleheavy or light chain variable regions. The single variable region can beused to screen for a second variable region capable of forming atwo-domain specific antigen-binding fragment capable of, for example,binding to TLR3. The screening may be accomplished by phage displayscreening methods using for example hierarchical dual combinatorialapproach disclosed in PCT Publ. No. WO92/01047. In this approach, anindividual colony containing either a H or L chain clone is used toinfect a complete library of clones encoding the other chain (L or H),and the resulting two-chain specific antigen-binding domain is selectedin accordance with phage display techniques as described.

TABLE 2a HCDR1 HCDR2 HCDR3 mAb CDR definition SEQ ID Sequence SEQ IDSequence SEQ ID Sequence 14 IMGT  82 GYSFTNYW  86 IDPSDSYTNY  84ARELYQGYMDTFDS 14 Kabat NYWVG FIDPSDSYTNYAPSFQ ELYQGYMDTFDS 14 ChothiaGYSFT PSDSYT LYQGYMDTFD 14 Consensus 111 GYSFTNYWVG 112 FIDPSDSYTNYAPSFQ 84 ARELYQGYMDTFDS 15 IMGT  82 GYSFTNYW  86 IDPSDSYTNY  84ARELYQGYMDTFDS 15 Kabat NYWVG FIDPSDSYTNYAPSFQ ELYQGYMDTFDS 15 ChothiaGYSFT PSDSYT LYQGYMDTFD 15 Consensus 111 GYSFTNYWVG 112 FIDPSDSYTNYAPSFQ 84 ARELYQGYMDTFDS 15-1 IMGT  82 GYSFTNYW  86 IDPSDSYTNY  84ARELYQGYMDTFDS 15-1 Kabat NYWVG RIDPSDSYTNYAPSFQ ELYQGYMDTFDS 15-1Chothia GYSFT PSDSYT LYQGYMDTFD 15-1 Consensus 111 GYSFTNYWVG 114RIDPSDSYTNYAPSFQ  84 ARELYQGYMDTFDS 15-2 IMGT  82 GYSFTNYW  86IDPSDSYTNY  84 ARELYQGYMDTFDS 15-2 Kabat NYWIG FIDPSDSYTNYAPSFQELYQGYMDTFDS 15-2 Chothia GYSFT PSDSYT LYQGYMDTFD 15-2 Consensus 115GYSFTNYWIG 112 FIDPSDSYTNYAPSFQ  84 ARELYQGYMDTFDS 15-3 IMGT  82GYSFTNYW  86 IDPSDSYTNY  84 ARELYQGYMDTFDS 15-3 Kabat NYWIS  86FIDPSDSYTNYAPSFQ  84 ELYQGYMDTFDS 15-3 Chothia GYSFT PSDSYT LYQGYMDTFD15-3 Consensus 116 GYSFTNYWIS 112 FIDPSDSYTNYAPSFQ  84 ARELYQGYMDTFDS15-4 IMGT  82 GYSFTNYW  86 IDPSDSYTNY  84 ARELYQGYMDTFDS 15-4 KabatNYWVG FIDPSDSYTNYSPSFQ ELYQGYMDTFDS 15-4 Chothia GYSFT PSDSYT LYQGYMDTFD15-4 Consensus 111 GYSFTNYWVG 117 FIDPSDSYTNYSPSFQ  84 ARELYQGYMDTFDS15-5 IMGT  82 GYSFTNYW  86 IDPSDSYTNY  84 ARELYQGYMDTFDS 15-5 KabatNYWIS RIDPSDSYTNYSPSFQ ELYQGYMDTFDS 15-5 Chothia GYSFT PSDSYT LYQGYMDTFD15-5 Consensus 116 GYSFTNYWIS 118 RIDPSDSYTNYSPSFQ  84 ARELYQGYMDTFDS15-6 IMGT  82 GYSFTNYW  86 IDPSDSYTNY ARQLYQGYMDTFDS 15-6 Kabat NYWISFIDPSDSYTNYAPSFQ QLYQGYMDTFDS 15-6 Chothia GYSFT PSDSYT LYQGYMDTFD 15-6Consensus 116 GYSFTNYWIS 112 FIDPSDSYTNYAPSFQ 119 ARQLYQGYMDTFDS 15-7IMGT  82 GYSFTNYW  86 IDPSDSYTNY  84 ARELYQGYMDTFDS 15-7 Kabat NYWVGFIDPSDSYTNYAPSFQ ELYQGYMDTFDS 15-7 Chothia GYSFT PSDSYT LYQGYMDTFD 15-7Consensus 111 GYSFTNYWVG 112 FIDPSDSYTNYAPSFQ  84 ARELYQGYMDTFDS 15-8IMGT  82 GYSFTNYW  86 IDPSDSYTNY  84 ARELYQGYMDTFDS 15-8 Kabat NYWVGFIDPSDSYTNYAPSFQ ELYQGYMDTFDS 15-8 Chothia GYSFT PSDSYT LYQGYMDTFD 15-8Consensus 111 GYSFTNYWVG 112 FIDPSDSYTNYAPSFQ  84 ARELYQGYMDTFDS 15-9IMGT  82 GYSFTNYW  86 IDPSDSYTNY 119 ARQLYQGYMDTFDS 15-9 Kabat NYWISRIDPSDSYTNYSPSFQG QLYQGYMDTFDS 15-9 Chothia GYSFT PSDSYT LYQGYMDTFD 15-9Consensus 116 GYSFTNYWIS 118 RIDPSDSYTNYSPSFQG 119 ARQLYQGYMDTFDS

In other embodiments, the invention provides an isolated antibody orfragment reactive with TLR3 comprising both a heavy chain variableregion and a light chain variable region having amino acid sequences atleast 95% identical to the variable region amino acid sequences shown inTable 3a.

In another aspect, the invention provides an isolated antibody havingcertain heavy chain and light chain amino acid sequences as shown inTable 3b.

Another aspect of the invention is isolated polynucleotides encoding anyof the antibodies of the invention or their complement. Certainexemplary polynucleotides are disclosed herein, however, otherpolynucleotides which, given the degeneracy of the genetic code or codonpreferences in a given expression system, encode the antibodyantagonists of the invention are also within the scope of the invention.

TABLE 2b LCDR1 LCDR2 LCDR3 SEQ ID SEQ ID SEQ ID mAb CDR definition NO:Sequence NO: Sequence NO: Sequence 14 IMGT  79 QSIGLY  80 AAS  85QQAETVSPT 14 Kabat RASQSIGLYLA AASSLQS QQAETVSPT 14 Chothia SQSIGLY AASAEIVSP 14 Consensus 109 RASQSIGLYLA 110 AASSLQS  85 QQAETVSPT 15 IMGT 79 QSIGLY  80 AAS  87 QQGNTLSYT 15 Kabat RASQSIGLYLA AASSLQS QQGNTLSYT15 Chothia SQSIGLY AAS GNTLSY 15 Consensus 109 RASQSIGLYLA 110 AASSLQS113 QQGNTLSYT 15-1 IMGT  79 QSIGLY  80 AAS  87 QQGNTLSYT 15-1 KabatRASQSIGLYLA AASSLQS QQGNTLSYT 15-1 Chothia SQSIGLY AAS GNTLSY 15-1Consensus 109 RASQSIGLYLA 110 AASSLQS 113 QQGNTLSYT 15-2 IMGT  79 QSIGLY 80 AAS  87 QQGNTLSYT 15-2 Kabat RASQSIGLYLA AASSLQS QQGNTLSYT 15-2Chothia SQSIGLY AAS GNTLSY 15-2 Consensus 109 RASQSIGLYLA 110 AASSLQS113 QQGNTLSYT 15-3 IMGT  79 QSIGLY  80 AAS  87 QQGNTLSYT 15-3 KabatRASQSIGLYLA AASSLQS QQGNTLSYT 15-3 Chothia SQSIGLY AAS GNTLSY 15-3Consensus 109 RASQSIGLYLA 110 AASSLQS 113 QQGNTLSYT 15-4 IMGT  79 QSIGLY 80 AAS  87 QQGNTLSYT 15-4 Kabat RASQSIGLYLA AASSLQS QQGNTLSYT 15-4Chothia SQSIGLY AAS GNTLSY 15-4 Consensus 109 RASQSIGLYLA 110 AASSLQS113 QQGNTLSYT 15-5 IMGT  79 QSIGLY  80 AAS  87 QQGNTLSYT 15-5 KabatRASQSIGLYLA AASSLQS QQGNTLSYT 15-5 Chothia SQSIGLY AAS GNTLSY 15-5Consensus 109 RASQSIGLYLA 110 AASSLQS 113 QQGNTLSYT 15-6 IMGT  79 QSIGLY 80 AAS  87 QQGNTLSYT 15-6 Kabat RASQSIGLYLA AASSLQS QQGNTLSYT 15-6Chothia SQSIGLY AAS GNTLSY 15-6 Consensus 109 RASQSIGLYLA 110 AASSLQS113 QQGNTLSYT 15-7 IMGT QSISSY  80 AAS  87 QQGNTLSYT 15-7 KabatRASQSISSYLA AASSLQS QQGNTLSYT 15-7 Chothia SQSISSY AAS GNTLSY 15-7Consensus 120 RASQSISSYLA 110 AASSLQS 113 QQGNTLSYT 15-8 IMGT  79 QSIGLY 80 AAS  87 QQGNTLSYT 15-8 Kabat RASQSIGLYLN AASSLQS QQGNTLSYT 15-8Chothia SQSIGLY AAS GNTLSY 15-8 Consensus 121 RASQSIGLYLN 110 AASSLQS113 QQGNTLSYT 15-9 IMGT  79 QSIGLY  80 AAS  87 QQGNTLSYT 15-9 KabatRASQSIGLYLA AASSLQS QQGNTLSYT 15-9 Chothia SQSIGLY AAS GNTLSY 15-9Consensus 109 RASQSIGLYLA 110 AASSLQS 113 QQGNTLSYT

TABLE 3a SEQ ID NO: mAb no: HV LV 16 6 5 17 8 7 18 10 9 19 12 11  1 1413  2 16 15  3 18 17  4 20 19  5 22 21  6 24 23  7 26 25  8 28 27  9 3029 10 32 31 11 34 33 12 36 35 13 38 37 14 40 39 15 42 41 15-1 124 4115-2 125 41 15-3 126 41 15-4 127 41 15-5 128 41 15-6 129 41 15-7 42 12215-8 42 123 15-9 159 41 15-10 129 225 F17 198 197 F18 200 199 F19 202201 c1811 164 163 9QVQ/QSV 212 209 10QVQ/QSV 213 210 12QVQ/QSV 214 21114EVQ 215 39 15EVQ 216 41

Exemplary antibody antagonists may be antibodies of the IgG, IgD, IgG,IgA or IgM isotypes. Additionally, such antibody antagonists can bepost-translationally modified by processes such as glycosylation,isomerization, deglycosylation or non-naturally occurring covalentmodification such as the addition of polyethylene glycol (PEG) moieties(pegylation) and lipidation. Such modifications may occur in vivo or invitro. For example, the antibodies of the invention can be conjugated topolyethylene glycol (PEGylated) to improve their pharmacokineticprofiles. Conjugation can be carried out by techniques known to thoseskilled in the art. Conjugation of therapeutic antibodies with PEG hasbeen shown to enhance pharmacodynamics while not interfering withfunction. (Deckert et al., Int. J. Cancer 87:382-390, 2000; Knight etal., Platelets 15:409-418, 2004; Leong et al., Cytokine 16:106-119,2001; Yang et al., Protein Eng. 16:761-770, 2003).

TABLE 3b Heavy chain Light chain mAb no: SEQ ID NO: SEQ ID NO: 14 102155 15 102 156 15-1 130 156 15-2 131 156 15-3 132 156 15-4 133 156 15-5134 156 15-6 135 156 15-7 102 157 15-8 102 158 15-9 160 156 15-10 135227 F17 204 203 F18 206 205 F19 208 207 14EVQ 220 155 15EVQ 220 156 5429166 165 c1811 168 167

Pharmacokinetic properties of the antibodies of the invention could alsobe enhanced through Fc modifications by techniques known to thoseskilled in the art. For example, IgG4 isotype heavy chains contain aCys-Pro-Ser-Cys (CPSC) motif in the hinge region capable of formingeither inter- or intra-heavy chain disulfide bonds, i.e., the two Cysresidues in the CPSC motif may disulfide bond with the corresponding Cysresidues in the other heavy chain (inter) or the two Cys residues withina given CPSC motif may disulfide bond with each other (intra). It isbelieved that in vivo isomerase enzymes are capable of convertinginter-heavy chain bonds of IgG4 molecules to intra-heavy chain bonds andvice versa (Aalberse and Schuurman, Immunology 105:9-19, 2002).Accordingly, since the heavy:light chain (H:L) pairs in those IgG4molecules with intra-heavy chain bonds in the hinge region are notcovalently associated with each other, they may dissociate into H:Lmonomers that then reassociate with H:L monomers derived from other IgG4molecules forming bispecific, heterodimeric IgG4 molecules. In abispecific IgG antibody the two Fabs of the antibody molecule differ inthe epitopes that they bind. Substituting the Ser residue in the hingeregion CPSC motif of IgG4 with Pro results in “IgG1-like behavior,”i.e., the molecules form stable disulfide bonds between heavy chains andtherefore, are not susceptible to H:L exchange with other IgG4molecules. In one embodiment, the antibodies of the invention willcomprise an IgG4 Fc domain with a S to P mutation in the CPSC motif. Thelocation of the CPSC motif is typically found at residue 228 of a matureheavy chain but can change depending on CDR lengths.

Further, sites can be removed that affect binding to Fc receptors otherthan an FcRn salvage receptor in the antibodies of the invention. Forexample, the Fc receptor binding regions involved in ADCC activity canbe removed in the antibodies of the invention. For example, mutation ofLeu234/Leu235 in the hinge region of IgG1 to L234A/L235A orPhe235/Leu236 in the hinge region of IgG4 to P235A/L236A minimizes FcRbinding and reduces the ability of the immunoglobulin to mediatecomplement dependent cytotoxicity and ADCC. In one embodiment, theantibodies of the invention will comprise an IgG4 Fc domain withP235A/L236A mutations. The location of these residues identified aboveis typical in a mature heavy chain but can change depending on CDRlengths. Exemplary antibodies having P235A/L236A mutations areantibodies having heavy chain amino acid sequences shown in SEQ ID NOs:218, 219 or 220.

Fully human, human-adapted, humanized and affinity-matured antibodymolecules or antibody fragments are within the scope of the invention asare fusion proteins and chimeric proteins. Antibody affinity towards anantigen may be improved by rational design or random affinity maturationusing well-known methods such as random or directed mutagenesis, oremploying phage display libraries. For example, substitutions can bemade to the Vernier Zone residues that mostly reside in the frameworkregion or to the “Affinity Determining Residues”, ADRs, to modulateaffinity of an antibody (U.S. Pat. No. 6,639,055; PCT Publ. No.WO10/045340).

Fully human, human-adapted, humanized, affinity-matured antibodymolecules or antibody fragments modified to improve stability,selectivity, cross-reactivity, affinity, immunogenicity or otherdesirable biological or biophysical property are within the scope of theinvention. Stability of an antibody is influenced by a number offactors, including (1) core packing of individual domains that affectstheir intrinsic stability, (2) protein/protein interface interactionsthat have impact upon the HC and LC pairing, (3) burial of polar andcharged residues, (4) H-bonding network for polar and charged residues;and (5) surface charge and polar residue distribution among other intra-and inter-molecular forces (Worn et al., J. Mol. Biol., 305:989-1010,2001). Potential structure destabilizing residues may be identifiedbased upon the crystal structure of the antibody or by molecularmodeling in certain cases, and the effect of the residues on antibodystability can be tested by generating and evaluating variants harboringmutations in the identified residues. One of the ways to increaseantibody stability is to raise the thermal transition midpoint (Tm) asmeasured by differential scanning calorimetry (DSC). In general, theprotein Tm is correlated with its stability and inversely correlatedwith its susceptibility to unfolding and denaturation in solution andthe degradation processes that depend on the tendency of the protein tounfold (Remmele et al., Biopharm., 13:36-46, 2000). A number of studieshave found correlation between the ranking of the physical stability offormulations measured as thermal stability by DSC and physical stabilitymeasured by other methods (Gupta et al., AAPS PharmSci. 5E8, 2003; Zhanget al., J. Pharm. Sci. 93:3076-3089, 2004; Maa et al., Int. J. Pharm.,140:155-168, 1996; Bedu-Addo et al., Pharm. Res., 21:1353-1361, 2004;Remmele et al., Pharm. Res., 15:200-208, 1997). Formulation studiessuggest that a Fab Tm has implication for long-term physical stabilityof a corresponding mAb. Differences in amino acids in either frameworkor within the antigen-binding sites could have significant effects onthe thermal stability of the Fab domain (Yasui, et al., FEBS Lett.353:143-146, 1994).

The antibody antagonists of the invention may bind TLR3 with a K_(d)less than or equal to about 10⁻⁷, 10⁻⁸, 10⁻⁹, 10⁻¹⁰, 10⁻¹¹ or 10⁻¹² M.The affinity of a given molecule for TLR3, such as an antibody can bedetermined experimentally using any suitable method. Such methods mayutilize Biacore or KinExA instrumentation, ELISA or competitive bindingassays known to those skilled in the art.

Antibody antagonists binding a given TLR3 homolog with a desiredaffinity can be selected from libraries of variants or fragments bytechniques including antibody affinity maturation.

Antibody antagonists can be identified based on their inhibition of TLR3biological activity using any suitable method. Such methods may utilizereporter-gene assays or assays measuring cytokine production using wellknown methods and as described in the application.

Another embodiment of the invention is a vector comprising at least onepolynucleotide of the invention. Such vectors may be plasmid vectors,viral vectors, vectors for baculovirus expression, transposon basedvectors or any other vector suitable for introduction of thepolynucleotides of the invention into a given organism or geneticbackground by any means.

Another embodiment of the invention is a host cell comprising any of thepolynucleotides of the invention such as a polynucleotide encoding apolypeptide comprising an immunoglobulin heavy chain variable regionhaving the amino acid sequence shown in SEQ ID NO: 6, 8, 10, 12, 14, 16,18, 20, 22, 24, 26, 28, 30, 32, 34, 36, 38, 40, 42, 124, 125, 126, 127,128, 129, 159, 198, 200, 202, 164, 212, 213, 214, 215 or 216 or animmunoglobulin light chain variable region having the amino acidsequence shown in SEQ ID NO: 5, 7, 9, 11, 13, 15, 17, 19, 21, 23, 25,27, 29, 31, 33, 35, 37, 39, 41, 122, 123, 197, 199, 201, 163, 209, 210,211, or 225.

Another embodiment of the invention is a host cell comprising apolynucleotide encoding a polypeptide comprising an immunoglobulin heavychain having the amino acid sequence shown in SEQ ID NO: 102, 130, 131,132, 133, 134, 135, 160, 204, 206, 208, 220, 166 or 168, or animmunoglobulin light chain having the amino acid sequence shown in SEQID NO: 155, 156, 157, 158, 203, 205, 207, 165, 167, or 227. Such hostcells may be eukaryotic cells, bacterial cells, plant cells or archealcells. Exemplary eukaryotic cells may be of mammalian, insect, avian orother animal origins. Mammalian eukaryotic cells include immortalizedcell lines such as hybridomas or myeloma cell lines such as SP2/0(American Type Culture Collection (ATCC), Manassas, Va., CRL-1581), NS0(European Collection of Cell Cultures (ECACC), Salisbury, Wiltshire, UK,ECACC No. 85110503), FO (ATCC CRL-1646) and Ag653 (ATCC CRL-1580) murinecell lines. An exemplary human myeloma cell line is U266 (ATTCCRL-TIB-196). Other useful cell lines include those derived from ChineseHamster Ovary (CHO) cells such as CHO-K1SV (Lonza Biologics,Walkersville, Md.), CHO-K1 (ATCC CRL-61) or DG44.

Another embodiment of the invention is a method of making an antibodyreactive with TLR3 comprising culturing a host cell of the invention andrecovering the antibody produced by the host cell. Methods of makingantibodies and purifying them are well known in the art.

Another embodiment of the invention is a hybridoma cell line thatproduces an antibody of the invention.

Another embodiment of the invention is an isolated antibody or fragmentthereof, wherein the antibody binds toll-like receptor 3 (TLR3) aminoacid residues K416, K418, L440, N441, E442, Y465, N466, K467, Y468,R488, R489, A491, K493, N515, N516, N517, H539, N541, 5571, L595, andK619 of SEQ ID NO: 2.

Another embodiment is an isolated antibody or fragment thereof, whereinthe antibody binds toll-like receptor 3 (TLR3) amino acid residues 5115,D116, K117, A120, K139, N140, N141, V144, K145, T166, Q167, V168, 5188,E189, D192, A195, and A219 of SEQ ID NO: 2.

Several well known methodologies can be employed to determine thebinding epitope of the antibodies of the invention. For example, whenthe structures of both individual components are known, in silicoprotein-protein docking can be carried out to identify compatible sitesof interaction. Hydrogen-deuterium (H/D) exchange can be carried outwith the antigen and antibody complex to map regions on the antigen thatmay be bound by the antibody. Segment and point mutagenesis of theantigen can be used to locate amino acids important for antibodybinding. For large proteins such as TLR3, point mutagenesis mapping issimplified when the binding site is first localized to a region on theprotein, such as by docking, segment mutagenesis or H/D exchange. Whenthe structures of both individual components are known, in silicoprotein-protein docking can be carried out to identify compatible sitesof interaction. Co-crystal structure of antibody-antigen complex can beused to identify residues contributing to the epitope and paratope.

Another embodiment of the invention is an isolated antibody or fragmentthereof, wherein the antibody binds TLR3 having an amino acid sequenceshown in SEQ ID NO: 2 with the heavy chain variable region Chothiaresidues W33, F50, D52, D54, Y56, N58, P61, E95, Y97, Y100, and D100b,and with the light chain variable region Chothia residues Q27, Y32, N92,T93, L94, and S95. The heavy chain paratope and the light chain paratopeChothia residues correspond to heavy chain residues W33, F50, D52, D55,Y57, N59, P62, E99, Y101, Y104, and D106 of SEQ ID NO: 216 and lightchain residues Q27, Y32, N92, T93, L94, and S95 of SEQ ID NO: 41.

Another embodiment of the invention is an isolated antibody or fragmentthereof, wherein the antibody binds TLR3 having an amino acid sequenceshown in SEQ ID NO: 2 with the heavy chain variable region Chothiaresidues N31a, Q52, R52b, S53, K54, Y56, Y97, P98, F99, and Y100, andwith the light chain variable region Chothia residues G29, S30, Y31,Y32, E50, D51, Y91, D92, and D93. The heavy chain paratope and the lightchain paratope Chothia residues correspond to heavy chain residues N32,Q54, R56, S57, K58, Y60, Y104, P105, F106, and Y107 of SEQ ID NO: 214and light chain residues G28, S29, Y30, Y31, E49, D50, Y90, D91, and D92of SEQ ID NO: 211.

Isolated antibodies having certain paratope residues that bind TLR3 canbe made by for example grafting the paratope residues into a suitablescaffold, assembling the engineered scaffolds into full antibodies,expressing the resulting antibodies, and testing the antibodies forbinding to TLR3 or for an effect on TLR3 biological activity. Exemplaryscaffolds are amino acid sequences of human antibody variable regionsencoded by human germline genes. The scaffolds can be selected based onfor example overall sequence homology, % identity between the paratoperesidues, or canonical structure class identity between the scaffold andan exemplary antibody, such as mAb 15EVQ or mAb 12QVQ/QSV. Humanantibody germline genes are disclosed in, for example, Tomlinson et al.,J. Mol. Biol 227:776-798, and at the International ImMunoGeneTics (IMGT)database (http_://_www_imgt_org). Consensus human framework regions canalso be used, e.g., as described in U.S. Pat. No. 6,300,064. Selectionof suitable scaffold can be done for example according to methodsdescribed in PCT Publ. No. WO10/045340.

Exemplary human germline genes that can be used as scaffolds onto whichthe paratope residues are grafted are the genes encoded by the Vκ1, Vλ3,Vh5, Vh6, Jκ, Jλ, and the Jh frameworks. The germline J-regions are usedin their entirety or in part to select FR4 sequences. For example, themAb 15EVQ light chain paratope residues can be grafted to a Vκ1framework encoded by IGKV1-39*01 that is joined directly to the J regionsequence encoded by IGKJ1. Sequences from other Vκ1 genes can also beused, and the FR4 sequences of other JK genes can be substituted inplace of IGKJ1. The mAb 15EVQ heavy chain paratope residues can begrafted to a Vh5 framework encoded by IGHV5-51*01, followed by about11-13 residues, for example 12 residues, constituting HCDR3 and the FR4sequence encoded by IGHJ1. The 11-13 residues span between the end ofthe FR3 region (“CAR”) and the start of the FR4 region (WGQ for most JHregions) and include 4 defined paratope residues from mAb 15EVQ Vh.Sequences from other Vh5 genes can also be used, and the FR4 sequencesof other Jh genes can be substituted in place of IGJH1. In anotherexample, the mAb 12QVQ/QSV light chain paratope residues can be graftedto a Vλ3 framework encoded by IGLV3-1*01 that is joined directly to theJ region sequence encoded by IGJL2. Sequences of other Vλ3 and Jλ genescan also be used. The length of LCDR3 is maintained at about 9-11residues, for example 10 residues. These about 9-11 residues spanbetween the end of the FR3 region (“YYC” for most V lambda scaffolds)and the start of the FR4 region (“FGG” for most JL regions) and include3 defined paratope residues from mAb 12QVQ/QSV. The mAb 12QVQ/QSV heavychain paratope residues can be grafted to a Vh6 framework encoded byIGHV6-1*01, followed by about 9-11 residues, for example 10 residues,constituting HCDR3, and the FR4 sequence encoded by IGJH1. The about9-11 residues span between the end of the FR3 region (“CAR”) and thestart of the FR4 region (WGQ for most JH regions) and include 4 definedparatope residues from mAb 12QVQ/QSV Vh. The FR4 sequences of other Jhgenes can be substituted in place of IGHJ1. The binding to TLR3 andbiological activity of the resulting antibody can be evaluated usingstandard methods. Alignments of the mAb 15EVQ and the mAb 12QVQ/QSVlight chain variable regions and heavy chain variable regions with theexemplary Vκ1, Vh5, Vλ3, Vh6, Jκ, Jλ or Jh genes are shown in FIGS.32-35. The paratope-grafted engineered antibodies can further bemodified by substitutions of the Vernier Zone residues or the AffinityDetermining Residues to improve antibody properties, for exampleaffinity, as described above. As long as the paratope-grafted antibodyretains binding to TLR3, the framework amino acid sequence in theparatope-grafted antibody may be 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%,98%, or 99% identical to the the mAb 15EVQ or 12QVQ/QSV frameworksequences.

Sequences from the antigen-binding sites can be grafted in addition tothe paratope residues using standard methods. For example, a completeHCDR3 or LCDR3 may be grafted.

Another aspect of the invention is an isolated antibody or fragmentthereof reactive with TLR3 that competes for TLR3 binding with amonoclonal antibody, wherein the monoclonal antibody comprises the aminoacid sequences of certain heavy chain complementarity determiningregions (CDRs) 1, 2 and 3, the amino acid sequences of certain lightchain CDRs 1, 2 and 3, the amino acid sequences of certain heavy chainvariable regions (VH) or the amino acid sequence of certain light chainvariable regions (VL). Examplary monoclonal antibodies of the inventionare an isolated antibody comprising a heavy chain variable region havingan amino acid sequence shown in SEQ ID NO: 216 and a light chainvariable region amino acid sequence shown in SEQ ID NO: 41, and anantibody comprising a heavy chain variable region having an amino acidsequence shown in SEQ ID NO: 214 and a light chain variable region aminoacid sequence shown in SEQ ID NO: 211.

Competition between binding to TLR3 can be assayed in vitro using wellknown methods. For example, binding of MSD Sulfo-Tag™ NHS-ester-labeledantibody to TLR3 in the presence of an unlableled antibody can beassessed by ELISA. Exemplary antibodies of the invention are mAb 12, mAb15 and mAb c1811 (see Table 3a). Previously described anti-TLR3antibodies c1068 and its derivatives (described in PCT Publ. No.WO06/060513A2), TLR3.7 (eBiosciences, cat no 14-9039) and ImgenexIMG-315A (Imgenex IMG-315A; generated against human TLR3 amino acidsamino acids 55-70, VLNLTHNQLRRLPAAN) do not compete with binding to TLR3with mAbs 12, 15 or c1811 as shown in Example 5.

Another aspect of the invention is an isolated antibody reactive withTLR3, wherein the antibody has at least one of the following properties:

-   -   a. binds to human TLR3 with a Kd of <10 nM;    -   b. reduces human TLR3 biological activity in an in vitro        poly(I:C) NF-kB reporter gene assay >50% at 1 μg/ml;    -   c. inhibits >60% of IL-6 or CXCL5/IP-10 production from BEAS-2B        cells stimulated with <100 ng/ml poly(I:C) at 10 μg/ml;    -   d. inhibits >50% of IL-6 or CXCL5/IP-10 production from BEAS-2B        cells stimulated with <100 ng/ml poly(I:C) at 0.4 μg/ml;    -   e. inhibits >50% of IL-6 production from NHBE cells stimulated        with 62.5 ng/ml poly(I:C) at 5 μg/ml;    -   f. inhibits >50% of IL-6 production from NHBE cells stimulated        with 62.5 ng/ml poly(I:C) at 1 μg/ml;    -   g. inhibits >20% of poly(I:C)-induced IFN-γ, IL-6 or IL-12        production by PBMC cells at 1 μg/ml.    -   h. inhibits cynomologus TLR3 biological activity in an in vitro        NF-kB reporter gene assay with IC50<10 μg/ml; or    -   i. inhibits cynomologus TLR3 biological activity in an in vitro        ISRE reporter gene assay with IC50<5 μg/ml.

Methods of Treatment

TLR3 antagonists of the invention, for example TLR3 antibodyantagonists, can be used to modulate the immune system. While notwishing to be bound by any particular theory, the antagonists of theinvention may modulate the immune system by preventing or reducingligand binding to TLR3, dimerization of TLR3, TLR3 internalization orTLR3 trafficking. The methods of the invention may be used to treat ananimal patient belonging to any classification. Examples of such animalsinclude mammals such as humans, rodents, dogs, cats and farm animals.For example, the antibodies of the invention are useful in antagonizingTLR3 activity, in the treatment of inflammation, inflammatory andmetabolic diseases and are also useful in the preparation of amedicament for such treatment wherein the medicament is prepared foradministration in dosages defined herein.

Generally, inflammatory conditions, infection-associated conditions orimmune-mediated inflammatory disorders that may be prevented or treatedby administration of the TLR3 antibody antagonists of the inventioninclude those mediated by cytokines or chemokines and those conditionswhich result wholly or partially from activation of TLR3 or signalingthrough the TLR3 pathway. Examples of such inflammatory conditionsinclude sepsis-associated conditions, inflammatory bowel diseases,autoimmune disorders, inflammatory disorders and infection-associatedconditions. It is also thought that cancers, cardiovascular andmetabolic conditions, neurologic and fibrotic conditions can beprevented or treated by administration of the TLR3 antibody antagonistsof the invention. Inflammation may affect a tissue or be systemic.Exemplary affected tissues are the respiratory tract, lung, thegastrointestinal tract, small intestine, large intestine, colon, rectum,the cardiovascular system, cardiac tissue, blood vessels, joint, boneand synovial tissue, cartilage, epithelium, endothelium, hepatic oradipose tissue. Exemplary systemic inflammatory conditions are cytokinestorm or hypercytokinemia, systemic inflammatory response syndrome(SIRS), graft versus host disease (GVHD), acute respiratory distresssyndrome (ARDS), severe acute respiratory distress syndrome (SARS),catastrophic anti-phospholipid syndrome, severe viral infections,influenza, pneumonia, shock, or sepsis.

Inflammation is a protective response by an organism to fend off aninvading agent. Inflammation is a cascading event that involves manycellular and humoral mediators. On one hand, suppression of inflammatoryresponses can leave a host immunocompromised; however, if leftunchecked, inflammation can lead to serious complications includingchronic inflammatory diseases (e.g. asthma, psoriasis, arthritis,rheumatoid arthritis, multiple sclerosis, inflammatory bowel disease andthe like), septic shock and multiple organ failure. Importantly, thesediverse disease states share common inflammatory mediators, such ascytokines, chemokines, inflammatory cells and other mediators secretedby these cells.

TLR3 activation by its ligands poly(I:C), dsRNA or endogenous mRNA leadsto activation of signaling pathways resulting in synthesis and secretionof pro-inflammatory cytokines, activation and recruitment ofinflammatory cells, such as macrophages, granulocytes, neutrophils andeosinophils, cell death, and tissue destruction. TLR3 induces secretionof IL-6, IL-8, IL-12, TNF-α, MIP-1, CXCL5/IP-10 and RANTES, and otherpro-inflammatory cytokines and chemokines implicated in immune cellrecruitment and activation, thus contributing to tissue destruction inautoimmune and other inflammatory diseases. TLR3 ligand endogenous mRNAis released from necrotic cells during inflammation, and may result in apositive feedback loop to activate TLR3 and perpetuate inflammation andfurther tissue damage. TLR3 antagonists, such as TLR3 antibodyantagonists, may normalize cytokine secretion, reduce recruitment ofinflammatory cells, and reduce tissue damage and cell death. Therefore,TLR3 antagonists have therapeutic potential to treat inflammation and aspectrum of inflammatory conditions.

One example of an inflammatory condition is sepsis-associated conditionthat may include systemic inflammatory response syndrome (SIRS), septicshock or multiple organ dysfunction syndrome (MODS). dsRNA released byviral, bacterial, fungal, or parasitic infection and by necrotic cellscan contribute to the onset of sepsis. While not wishing to be bound byan particular theory, it is believed that treatment with TLR3antagonists can provide a therapeutic benefit by extending survivaltimes in patients suffering from sepsis-associated inflammatoryconditions or prevent a local inflammatory event (e.g., in the lung)from spreading to become a systemic condition, by potentiating innateantimicrobial activity, by demonstrating synergistic activity whencombined with antimicrobial agents, by minimizing the local inflammatorystate contributing to the pathology, or any combination of theforegoing. Such intervention may be sufficient to permit additionaltreatment (e.g., treatment of underlying infection or reduction ofcytokine levels) necessary to ensure patient survival. Sepsis can bemodeled in animals, such as mice, by the administration ofD-galactosamine and poly(I:C). In such models, D-galactosamine is ahepatotoxin which functions as a sepsis sensitizer and poly(I:C) is asepsis-inducing molecule that mimics dsRNA and activates TLR3. TLR3antagonist treatment may increase animal survival rates in a murinemodel of sepsis, and thus TLR3 antagonists may be useful in thetreatment of sepsis.

Gastrointestinal inflammation is inflammation of a mucosal layer of thegastrointestinal tract, and encompasses acute and chronic inflammatoryconditions. Acute inflammation is generally characterized by a shorttime of onset and infiltration or influx of neutrophils. Chronicinflammation is generally characterized by a relatively longer period ofonset and infiltration or influx of mononuclear cells. Mucosal layer maybe mucosa of the bowel (including the small intestine and largeintestine), rectum, stomach (gastric) lining, or oral cavity. Exemplarychronic gastrointestinal inflammatory conditions are inflammatory boweldisease (IBD), colitis induced by environmental insults (e.g.,gastrointestinal inflammation (e.g., colitis) caused by or associatedwith (e.g., as a side effect) a therapeutic regimen, such asadministration of chemotherapy, radiation therapy, and the like),infections colitis, ischemic colitis, collagenous or lymphocyticcolitis, necrotizing enterocolitis, colitis in conditions such aschronic granulomatous disease or celiac disease, food allergies,gastritis, infectious gastritis or enterocolitis (e.g., Helicobacterpylori-infected chronic active gastritis) and other forms ofgastrointestinal inflammation caused by an infectious agent.

Inflammatory bowel disease (IBD) includes a group of chronicinflammatory disorders of generally unknown etiology, e.g., ulcerativecolitis (UC) and Crohn's disease (CD). Clinical and experimentalevidence suggest that the pathogenesis of IBD is multifactorialinvolving susceptibility genes and environmental factors. Ininflammatory bowel disease, the tissue damage results from aninappropriate or exaggerated immune response to antigens of the gutmicroflora. Several animal models for inflammatory bowel diseases exist.Some of the most widely used models are the 2,4,6-trinitrobenesulfonicacid/ethanol (TNBS)-induced colitis model or the oxazalone model, whichinduce chronic inflammation and ulceration in the colon (Neurath et al.,Intern. Rev. Immunol 19:51-62, 2000). Another model uses dextran sulfatesodium (DSS), which induces an acute colitis manifested by bloodydiarrhea, weight loss, shortening of the colon and mucosal ulcerationwith neutrophil infiltration. DSS-induced colitis is characterizedhistologically by infiltration of inflammatory cells into the laminapropria, with lymphoid hyperplasia, focal crypt damage, and epithelialulceration (Hendrickson et al., Clinical Microbiology Reviews 15:79-94,2002). Another model involves the adoptive transfer of naïveCD45RB^(high) CD4 T cells to RAG or SCID mice. In this model, donornaïve T cells attack the recipient gut causing chronic bowelinflammation and symptoms similar to human inflammatory bowel diseases(Read and Powrie, Curr. Protoc. Immunol. Chapter 15 unit 15.13, 2001).The administration of antagonists of the present invention in any ofthese models can be used to evaluate the potential efficacy of thoseantagonists to ameliorate symptoms and alter the course of diseasesassociated with inflammation in the gut, such as inflammatory boweldisease. Several treatment options for IBD are available, for exampleanti-TNF-α antibody therapies have been used for a decade to treatCrohn's disease (Van Assche et al., Eur. J. Pharmacol. Epub October2009). However, a significant percentage of patients are refractory tothe current treatments (Hanauer et al., Lancet 359:1541-1549, 2002;Hanauer et al., Gastroenterology 130:323-333, 2006), and thus newtherapies targeting refractory patient populations are needed.

Another example of an inflammatory condition is an inflammatorypulmonary condition. Exemplary inflammatory pulmonary conditions includeinfection-induced pulmonary conditions including those associated withviral, bacterial, fungal, parasite or prion infections; allergen-inducedpulmonary conditions; pollutant-induced pulmonary conditions such asasbestosis, silicosis, or berylliosis; gastric aspiration-inducedpulmonary conditions, immune dysregulation, inflammatory conditions withgenetic predisposition such as cystic fibrosis, and physicaltrauma-induced pulmonary conditions, such as ventilator injury. Theseinflammatory conditions also include asthma, emphysema, bronchitis,chronic obstructive pulmonary disease (COPD), sarcoidosis,histiocytosis, lymphangiomyomatosis, acute lung injury, acuterespiratory distress syndrome, chronic lung disease, bronchopulmonarydysplasia, community-acquired pneumonia, nosocomial pneumonia,ventilator-associated pneumonia, sepsis, viral pneumonia, influenzainfection, parainfluenza infection, rotavirus infection, humanmetapneumovirus infection, respiratory syncitial virus infection andaspergillus or other fungal infections. Exemplary infection-associatedinflammatory diseases may include viral or bacterial pneumonia,including severe pneumonia, cystic fibrosis, bronchitis, airwayexacerbations and acute respiratory distress syndrome (ARDS). Suchinfection-associated conditions may involve multiple infections such asa primary viral infection and a secondary bacterial infection.

Asthma is an inflammatory disease of the lung that is characterized byairway hyperresponsiveness (“AHR”), bronchoconstriction, wheezing,eosinophilic or neutrophilic inflammation, mucus hypersecretion,subepithelial fibrosis, and elevated IgE levels. Patients with asthmaexperience “exacerbations”, a worsening of symptoms, most commonly dueto microbial infections of the respiratory tract (e.g. rhinovirus,influenza virus, Haemophilus influenza, etc.). Asthmatic attacks can betriggered by environmental factors (e.g. ascarids, insects, animals(e.g., cats, dogs, rabbits, mice, rats, hamsters, guinea pigs andbirds), fungi, air pollutants (e.g., tobacco smoke), irritant gases,fumes, vapors, aerosols, chemicals, pollen, exercise, or cold air. Apartfrom asthma, several chronic inflammatory diseases affecting the lungare characterized by neutrophil infiltration to the airways, for examplechronic obstructive pulmonary disease (COPD), bacterial pneumonia andcystic fibrosis (Linden et al., Eur. Respir. J. 15:973-977, 2000; Rahmanet al., Clin. Immunol. 115:268-276, 2005), and diseases such as COPD,allergic rhinitis, and cystic fibrosis are characterized by airwayhyperresponsiveness (Fahy and O'Byrne, Am. J. Respir. Crit. Care Med.163:822-823, 2001). Commonly used animal models for asthma and airwayinflammation include the ovalbumin challenge model and methacholinesensitization models (Hessel et al., Eur. J. Pharmacol. 293:401-412,1995). Inhibition of cytokine and chemokine production from culturedhuman bronchial epithelial cells, bronchial fibroblasts or airway smoothmuscle cells can also be used as in vitro models. The administration ofantagonists of the present invention to any of these models can be usedto evaluate the use of those antagonists to ameliorate symptoms andalter the course of asthma, airway inflammation, COPD and the like.

Other inflammatory conditions and neuropathies, which may be preventedor treated by the methods of the invention are those caused byautoimmune diseases. These conditions and neuropathies include multiplesclerosis, systemic lupus erythematous, and neurodegenerative andcentral nervous system (CNS) disorders including Alzheimer's disease,Parkinson's disease, Huntington's disease, bipolar disorder andAmyotrophic Lateral Sclerosis (ALS), liver diseases including primarybiliary cirrhosis, primary sclerosing cholangitis, non-alcoholic fattyliver disease/steatohepatitis, fibrosis, hepatitis C virus (HCV) andhepatitis B virus (HBV), diabetes and insulin resistance, cardiovasculardisorders including atherosclerosis, cerebral hemorrhage, stroke andmyocardial infarction, arthritis, rheumatoid arthritis, psoriaticarthritis and juvenile rheumatoid arthritis (JRA), osteoporosis,osteoarthritis, pancreatitis, fibrosis, encephalitis, psoriasis, Giantcell arteritis, ankylosing spondolytis, autoimmune hepatitis, humanimmunodeficiency virus (HIV), inflammatory skin conditions, transplant,cancer, allergies, endocrine diseases, wound repair, other autoimmunedisorders, airway hyperresponsiveness and cell, virus, or prion-mediatedinfections or disorders.

Arthritis, including osteoarthritis, rheumatoid arthritis, arthriticjoints as a result of injury, and the like, are common inflammatoryconditions which would benefit from the therapeutic use ofanti-inflammatory proteins, such as the antagonists of the presentinvention. For example, rheumatoid arthritis (RA) is a systemic diseasethat affects the entire body and is one of the most common forms ofarthritis. Since rheumatoid arthritis results in tissue damage, TLR3ligands could be present at the site of the inflammation. Activation ofTLR3 signaling may perpetuate inflammation and further tissue damage inthe inflamed joint. Several animal models for rheumatoid arthritis areknown in the art. For example, in the collagen-induced arthritis (CIA)model, mice develop chronic inflammatory arthritis that closelyresembles human rheumatoid arthritis. Administration of the TLR3antagonists of the present invention to the CIA model mice can be usedto evaluate the use of these antagonists to ameliorate symptoms andalter the course of diseases.

Diabetes mellitus, diabetes, refers to a disease process derived frommultiple causative factors and characterized by hyperglycemia (LeRoithet al., (eds.), Diabetes Mellitus, Lippincott-Raven Publishers,Philadelphia, Pa. U.S.A. 1996), and all references cited therein.Uncontrolled hyperglycemia is associated with increased and prematuremortality due to an increased risk for microvascular and macrovasculardiseases, including nephropathy, neuropathy, retinopathy, hypertension,cerebrovascular disease and coronary heart disease. Therefore, controlof glucose homeostasis is a critically important approach for thetreatment of diabetes.

Underlying defects lead to a classification of diabetes into two majorgroups: type I diabetes (insulin dependent diabetes mellitus, IDDM),which arises when patients lack insulin-producing beta-cells in theirpancreatic glands, and type 2 diabetes (non-insulin dependent diabetesmellitus, NIDDM), which occurs in patients with an impaired beta-cellinsulin secretion and/or resistance to insulin action.

Type 2 diabetes is characterized by insulin resistance accompanied byrelative, rather than absolute, insulin deficiency. In insulin resistantindividuals, the body secretes abnormally high amounts of insulin tocompensate for this defect. When inadequate amounts of insulin arepresent to compensate for insulin resistance and adequately controlglucose, a state of impaired glucose tolerance develops. In asignificant number of individuals, insulin secretion declines furtherand the plasma glucose level rises, resulting in the clinical state ofdiabetes. Adipocity-associated inflammation has been strongly implicatedin the development of insulin resistance, type 2 diabetes, dyslipidemiaand cardiovascular disease. Obese adipose recruits and retainsmacrophages and can produce excessive pro-inflammatory cytokinesincluding TNF-α and IL-6, free fatty acids and adipokines, which caninterfere with insulin signaling and induce insulin resistance. TLR3activation on macrophages may contribute to the pro-inflammatory statusof the adipose. Several animal modes of insulin resistance are known.For example, in a diet-induced obesity model (DIO) animals develophyperglycemia and insulin resistance accompanied by weight gain.Administration of TLR3 antagonists of the present invention to the DIOmodel can be used to evaluate the use of the antagonists to amelioratecomplications associated with type 2 diabetes and alter the course ofthe disease.

Exemplary cancers may include at least one malignant disease in a cell,tissue, organ, animal or patient, including, but not limited toleukemia, acute leukemia, acute lymphoblastic leukemia (ALL), B-cell orT-cell ALL, acute myeloid leukemia (AML), chronic myelocytic leukemia(CML), chronic lymphocytic leukemia (CLL), hairy cell leukemia,myelodyplastic syndrome (MDS), a lymphoma, Hodgkin's disease, amalignant lymphoma, non-Hodgkin's lymphoma, Burkitt's lymphoma, multiplemyeloma, Kaposi's sarcoma, colorectal carcinoma, pancreatic carcinoma,renal cell carcinoma, breast cancer, nasopharyngeal carcinoma, malignanthistiocytosis, paraneoplastic syndrome/hypercalcemia of malignancy,solid tumors, adenocarcinomas, squamous cell carcinomas, sarcomas,malignant melanoma, particularly metastatic melanoma, hemangioma,metastatic disease, cancer related bone resorption and cancer relatedbone pain.

Exemplary cardiovascular diseases may include cardiovascular disease ina cell, tissue, organ, animal, or patient, including, but not limitedto, cardiac stun syndrome, myocardial infarction, congestive heartfailure, stroke, ischemic stroke, hemorrhage, arteriosclerosis,atherosclerosis, restenosis, diabetic atherosclerotic disease,hypertension, arterial hypertension, renovascular hypertension, syncope,shock, syphilis of the cardiovascular system, heart failure, corpulmonale, primary pulmonary hypertension, cardiac arrhythmias, atrialectopic beats, atrial flutter, atrial fibrillation (sustained orparoxysmal), post perfusion syndrome, cardiopulmonary bypassinflammation response, chaotic or multifocal atrial tachycardia, regularnarrow QRS tachycardia, specific arrhythmias, ventricular fibrillation,His bundle arrhythmias, atrioventricular block, bundle branch block,myocardial ischemic disorders, coronary artery disease, angina pectoris,myocardial infarction, cardiomyopathy, dilated congestivecardiomyopathy, restrictive cardiomyopathy, valvular heart diseases,endocarditis, pericardial disease, cardiac tumors, aordic and peripheralaneurysms, aortic dissection, inflammation of the aorta, occulsion ofthe abdominal aorta and its branches, peripheral vascular disorders,occulsive arterial disorders, peripheral atherosclerotic disease,thromboangitis obliterans, functional peripheral arterial disorders,Raynaud's phenomenon and disease, acrocyanosis, erythromelalgia, venousdiseases, venous thrombosis, varicose veins, arteriovenous fistula,lymphederma, lipedema, unstable angina, reperfusion injury, post pumpsyndrome and ischemia-reperfusion injury.

Exemplary neurological diseases may include neurologic disease in acell, tissue, organ, animal or patient, including, but not limited toneurodegenerative diseases, multiple sclerosis, migraine headache, AIDSdementia complex, demyelinating diseases, such as multiple sclerosis andacute transverse myelitis; extrapyramidal and cerebellar disorders suchas lesions of the corticospinal system; disorders of the basal gangliaor cerebellar disorders; hyperkinetic movement disorders such asHuntington's Chorea and senile chorea; drug-induced movement disorders,such as those induced by drugs which block CNS dopamine receptors;hypokinetic movement disorders, such as Parkinson's disease; Progressivesupranucleo Palsy; structural lesions of the cerebellum; spinocerebellardegenerations, such as spinal ataxia, Friedreich's ataxia, cerebellarcortical degenerations, multiple systems degenerations (Mencel,Dejerine-Thomas, Shi-Drager, and Machado-Joseph); systemic disorders(Refsum's disease, abetalipoprotemia, ataxia, telangiectasia, andmitochondrial multisystem disorder); demyelinating core disorders, suchas multiple sclerosis, acute transverse myelitis; and disorders of themotor unit such as neurogenic muscular atrophies (anterior horn celldegeneration, such as amyotrophic lateral sclerosis, infantile spinalmuscular atrophy and juvenile spinal muscular atrophy); Alzheimer'sdisease; Down's Syndrome in middle age; Diffuse Lewy body disease;Senile Dementia of Lewy body type; Wernicke-Korsakoff syndrome; chronicalcoholism; Creutzfeldt-Jakob disease; Subacute sclerosingpanencephalitis, Hallerrorden-Spatz disease and Dementia pugilistica.

Exemplary fibrotic conditions may include liver fibrosis (including butnot limited to alcohol-induced cirrhosis, viral-induced cirrhosis,autoimmune-induced hepatitis); lung fibrosis (including but not limitedto scleroderma, idiopathic pulmonary fibrosis); kidney fibrosis(including but not limited to scleroderma, diabetic nephritis,glomerular nehpritis, lupus nephritis); dermal fibrosis (including butnot limited to scleroderma, hypertrophic and keloid scarring, burns);myelofibrosis; neurofibromatosis; fibroma; intestinal fibrosis; andfibrotic adhesions resulting from surgical procedures. In such a method,the fibrosis can be organ specific fibrosis or systemic fibrosis. Theorgan specific fibrosis can be associated with at least one of lungfibrosis, liver fibrosis, kidney fibrosis, heart fibrosis, vascularfibrosis, skin fibrosis, eye fibrosis, bone marrow fibrosis or otherfibrosis. The lung fibrosis can be associated with at least one ofidiopathic pulmonary fibrosis, drug induced pulmonary fibrosis, asthma,sarcoidosis or chronic obstructive pulmonary disease. The liver fibrosiscan be associated with at least one of cirrhosis, schistomasomiasis orcholangitis. The cirrhosis can be selected from alcoholic cirrhosis,post-hepatitis C cirrhosis, primary biliary cirrhosis. The cholangitisis sclerosing cholangitis. The kidney fibrosis can be associated withdiabetic nephropathy or lupus glomeruloschelerosis. The heart fibrosiscan be associated with myocardial infarction. The vascular fibrosis canbe associated with postangioplasty arterial restenosis oratherosclerosis. The skin fibrosis can be associated with burn scarring,hypertrophic scarring, keloid, or nephrogenic fibrosing dermatopathy.The eye fibrosis can be associated with retro-orbital fibrosis,postcataract surgery or proliferative vitreoretinopathy. The bone marrowfibrosis can be associated with idiopathic myelofibrosis or drug inducedmyelofibrosis. The other fibrosis can be selected from Peyronie'sdisease, Dupuytren's contracture or dermatomyositis. The systemicfibrosis can be systemic sclerosis or graft versus host disease.

Administration/Pharmaceutical Compositions

The “therapeutically effective amount” of the agent effective in thetreatment or prevention of conditions where suppression of TLR3 activityis desirable can be determined by standard research techniques. Forexample, the dosage of the agent that will be effective in the treatmentor prevention of inflammatory condition such as asthma, Crohn's Disease,ulcerative colitis or rheumatoid arthritis can be determined byadministering the agent to relevant animal models, such as the modelsdescribed herein.

In addition, in vitro assays can optionally be employed to help identifyoptimal dosage ranges. Selection of a particular effective dose can bedetermined (e.g., via clinical trials) by those skilled in the art basedupon the consideration of several factors. Such factors include thedisease to be treated or prevented, the symptoms involved, the patient'sbody mass, the patient's immune status and other factors known by theskilled artisan. The precise dose to be employed in the formulation willalso depend on the route of administration, and the severity of disease,and should be decided according to the judgment of the practitioner andeach patient's circumstances. Effective doses can be extrapolated fromdose-response curves derived from in vitro or animal model test systems.

In the methods of the invention, the TLR3 antagonist may be administeredsingly or in combination with at least one other molecule. Suchadditional molecules may be other TLR3 antagonist molecules or moleculeswith a therapeutic benefit not mediated by TLR3 receptor signaling.Antibiotics, antivirals, palliatives and other compounds that reducecytokine levels or activity are examples of such additional molecules.

The mode of administration for therapeutic use of the agent of theinvention may be any suitable route that delivers the agent to the host.Pharmaceutical compositions of these agents are particularly useful forparenteral administration, e.g., intradermal, intramuscular,intraperitoneal, intravenous, subcutaneous or intranasal.

The agent of the invention may be prepared as pharmaceuticalcompositions containing an effective amount of the agent as an activeingredient in a pharmaceutically acceptable carrier. The term “carrier”refers to a diluent, adjuvant, excipient, or vehicle with which theactive compound is administered. Such pharmaceutical vehicles can beliquids, such as water and oils, including those of petroleum, animal,vegetable or synthetic origin, such as peanut oil, soybean oil, mineraloil, sesame oil and the like. For example, 0.4% saline and 0.3% glycinecan be used. These solutions are sterile and generally free ofparticulate matter. They may be sterilized by conventional, well-knownsterilization techniques (e.g., filtration). The compositions maycontain pharmaceutically acceptable auxiliary substances as required toapproximate physiological conditions such as pH adjusting and bufferingagents, stabilizing, thickening, lubricating and coloring agents, etc.The concentration of the agent of the invention in such pharmaceuticalformulation can vary widely, i.e., from less than about 0.5%, usually ator at least about 1% to as much as 15 or 20% by weight and will beselected primarily based on required dose, fluid volumes, viscosities,etc., according to the particular mode of administration selected.

Thus, a pharmaceutical composition of the invention for intramuscularinjection could be prepared to contain 1 ml sterile buffered water, andbetween about 1 ng to about 100 mg, e.g. about 50 ng to about 30 mg ormore preferably, about 5 mg to about 25 mg, of a TLR3 antibodyantagonist of the invention. Similarly, a pharmaceutical composition ofthe invention for intravenous infusion could be made up to contain about250 ml of sterile Ringer's solution, and about 1 mg to about 30 mg andpreferably 5 mg to about 25 mg of an antagonist of the invention. Actualmethods for preparing parenterally administrable compositions are wellknown and are described in more detail in, for example, “Remington'sPharmaceutical Science”, 15th ed., Mack Publishing Company, Easton, Pa.

The antibody antagonists of the invention can be lyophilized for storageand reconstituted in a suitable carrier prior to use. This technique hasbeen shown to be effective with conventional immunoglobulins and proteinpreparations and art-known lyophilization and reconstitution techniquescan be employed.

The present invention will now be described with reference to thefollowing specific, non-limiting examples.

Example 1 Identification and Derivation of Anti-huTLR3 Antagonist mAbs

The MorphoSys Human Combinatorial Antibody Library (HuCAL®) Gold phagedisplay library (Morphosys AG, Martinsried, Germany) was used as asource of human antibody fragments and was panned against a purifiedTLR3 antigen generated from the expression of amino acids 1-703 of humanTLR3 (huTLR3) (SEQ ID NO: 4) with a C-terminal poly-histidine tag andpurified by immobilized metal affinity chromatography. Amino acids 1-703correspond to the predicted extracellular domain (ECD) of huTLR3. Fabfragments (Fabs) that bound specifically to huTRL3 ECD were selected bypresenting the TLR3 protein in a variety of ways so that a diverse setof antibody fragments could be identified, sequenced and confirmed asunique. From different panning strategies, 62 candidates (differentV-region sequences) were identified as unique hTLR3 ECD binders.

The 62 candidates identified as huTLR3 ECD binders were screened forneutralizing activity in a range of cell-based assays relevant toidentifying anti-inflammatory activity. Using preliminary activity data(see Example 2 below), four candidates (Fabs 16-19) defining families16-19 were selected from the 62 as parents for CDR maturation of heavychain CDR2 (HCDR2) and light chain CDR3 (LCDR3). One of the parentalcandidates (candidate 19) exhibited an N-linked glycosylation site inHCDR2; a Ser to Ala (S to A) mutation was made in this candidate todelete the site. Following CDR maturation of the four parentalcandidates, a total of 15 progeny candidates (candidates 1-15) wereidentified for further characterization as described in Example 2 below.A listing of the light and heavy chain variable regions present in eachof the 19 candidates is shown in Table 3 above. The candidates areherein referred to as mAbs 1-19 or Fabs 1-19, depending whether theywere Fabs or cloned as full length antibody chains (Example 3). Due toexpression vector design, the mature amino termini of the variableregions for all candidates were QVE for heavy chain and DI for the lightchain.

The preferred sequences at these termini are those in the respectivegermline genes with high identity to the candidate sequences. Forfamilies 17 and 18 the germline sequences are QVQ for VH and SY for VL.For family 19, the sequences are EVQ for VH and DI for VL. The SYsequence is unique to the lambda subgroup 3 and there are reports ofheterogeneity with either S or Y as the amino terminal residue. Thus,the QSV consensus terminus from the prominent lambda subgroup 1 wasconsidered a more suitable replacement for DIE for VL of families 17 and18. These changes were introduced into candidates 9, 10 and 12 fromfamily 18 and candidates 14 and 15 from family 19. In this process, boththe VH and VL regions of these antibodies were codon optimized. Theamino acid sequences of the light chain variable region N-terminalgermline variants of candidates 9, 10 and 11 are shown in SEQ ID NO:s209-211, and the amino acid sequences of the heavy chain variable regionN-terminal germline variants for candidates 9, 10, 12, 14, and 15 areshown in SEQ ID NO:s 212-216, respectively. The N-terminal variants ofthe candidates are herein referred to as candidate/mAb/Fab 9QVQ/QSV,10QVQ/QSV, 12QVQ/QSV, 14EVQ or 15EVQ. The N-terminal germline variantswere expressed as mAbs and showed no effect on binding to TLR3 or intheir ability to inhibit TLR3 biological activity when compared to theirparent counterparts (data not shown).

Example 2 Determination of TLR3 Antagonist Activity In Vitro

The 15 CDR-matured candidates described above were selected as potentialhuman therapeutics and a range of binding and neutralizing activitieswere determined. The activity assays and results for the four parentalFabs, Fabs 16-19 and 15 CDR-matured Fabs, Fabs 1-15 or theirnon-germline V-region variants are described below.

Inhibition of NF-κB and ISRE Signaling Cascasde

293T cells were grown in DMEM and GlutaMax media (Invitrogen, Carlsbad,Calif.) supplemented with heat-inactivated FBS and transfected with 30ng pNF-κB or ISRE firefly luciferase reporter plasmids, 13.5 ng pcDNA3.1vector, 5 ng phRL-TK, and 1.5 ng pCDNA encoding FL TLR3 (SEQ ID NO: 2).The phRL-TK plasmid contains the Renilla luciferase gene driven by theHSV-1 thymidine kinase promoter (Promega, Madion, Wis.). TLR3 antibodieswere incubated 30-60 min. before addition of poly(I:C) (GE Healthcare,Piscataway, N.J.). The plates were incubated 6 h or 24 h at 37° C.before the addition of the Dual-Glo luciferase reagent, and the plateswere read on a FLUOstar plate reader. Normalized values (luciferaseratios) were obtained by dividing the firefly RLUs by the Renilla RLUs.Upon stimulation with the TLR3 agonist poly (I:C) (1 μg/ml), the NF-κBor ISRE signaling cascade stimulated firefly luciferase production wasspecifically inhibited by incubation of the cells with anti-TLR3antibodies (0.4, 2.0 and 10 μg/ml) prior to stimulation. The results forthe NF-κB assays are shown in FIG. 1 and are expressed as % inhibitionof the Firefly/Renilla ratio with 5465 as the positive control(neutralizing anti-human TLR3 Mab) and an anti-human tissue factor mAb(859) as the human IgG4 isotype control. >50% inhibition was achievedwith mAb concentrations 0.4-10 μg/ml. c1068 and TLR3.7 inhibited about38% and 8% of TLR3 biological activity at 10 μg/ml. Similar results wereobtained with the ISRE reporter gene assay (data not shown).

Cytokine Release in BEAS-2B Cells

BEAS-2B cells (SV-40 transformed normal human bronchial epithelial cellline) were seeded in a collagen type I coated dishes and incubated withor without anti-human TLR3 antibodies prior to addition of poly (I:C).Twenty-four hours after treatments, supernatants were collected andassayed for cytokine and chemokine levels using a custom multi-plex beadassay for detection of IL-6, IL-8, CCL-2/MCP-1, CCL5/RANTES, andCXCL10/IP-10. Results are shown in FIGS. 2A and 2B as % inhibition ofthe individual cytokine/chemokine following mAb treatment at 0.4, 2.0and 10 μg/ml. 5465 is a positive control; 859 is an isotype control.

Cytokine Release in NHBE Cells

Cytokine release was also assayed in normal human bronchial epithelial(NHBE) cells (Lonza, Walkersville, Md.). NHBE cells were expanded andtransferred to collagen-coated dishes and incubated for 48 hours afterwhich the media was removed and replenished with 0.2 ml of fresh media.The cells were then incubated with or without anti-human TLR3 mAbs 60minutes prior to the addition of poly (I:C). Supernatants were collectedafter 24 hours and stored at −20° C. or assayed immediately for IL-6levels. Results are graphed in FIGS. 3A and 3B as % inhibition of IL-6secretion following mAb treatment using doses between 0.001 and 50μg/ml. 5465 is a positive control, 859 is an isotype control. Most mAbsinhibited at least 50% of IL-6 production at <1 μg/ml, and achieved 75%inhibition at <5 μg/ml.

Cytokine Release in PBMC Cells

Cytokine release was also assayed in human peripheral blood mononuclearcells (PBMC). Whole blood was collected from human donors into heparincollection tubes to which a Ficoll-Paque Plus solution was slowlylayered underneath. The tubes were centrifuged and the PBMCs, thatformed a white layer just above the Ficoll, were recovered and plated.The PBMCs were then incubated with or without anti-human TLR3 mAbs priorto the addition of 25 μg/ml poly(I:C). After 24 hrs, supernatants werecollected and cytokine levels were determined using Luminex technology.Results are graphed in FIG. 4 as cumulative percentage inhibition ofIFN-γ, IL-12 and IL-6 using a single dose of mAb (0.4 μg/ml) with 5465is a positive control; hIgG4 is an isotype control.

Cytokine Release in HASM Cells

Briefly, human airway smooth muscle (HASM) cells were incubated with orwithout anti-human TLR3 mAbs prior to the addition of a synergisticcombination of 500 ng/ml poly(I:C) and 10 ng/ml INF-α. After 24 hrs,supernatants were collected and cytokine levels were determined usingLuminex technology. Results are graphed in FIGS. 5A and 5B as levels ofthe chemokine CCL5/RANTES using three doses of mAb (0.4, 2 and 10μg/ml). 5465 is a positive control; hIgG4 is an isotype control.

The results from the in vitro assays in human cells confirm the abilityof the antibodies of the invention to reduce cytokine and chemokinesrelease as a result of binding to huTLR3.

Example 3 Full-Length Antibody Constructs

The four parental Fabs (candidate nos. 16-19) and 15 progeny Fabs(candidate nos. 1-15) heavy chains were cloned onto a human IgG4background with a S229P Fc mutation. Candidates 9QVQ/QSV, 10QVQ/QSV,12QVQ/QSV, 14EVQ or 15EVQ were cloned onto a human IgG4 background withF235A/L236A and S229P Fc mutations.

The mature full-length heavy chain amino acid sequences are shown in SEQID NOs: 90-102 and 218-220 as follows:

Candidate SEQ ID NO: 16  90 17  91 18  92 19  93 1 94 2 95 3 96 4 97 5,6, 7 98 8 99 9 100 10, 11, 12 101 13, 14, 15 102 9EVQ 218 10EVQ, 12EVQ219 14EVQ, 15EVQ 220

For expression, these heavy chain sequences can include an N-terminalleader sequence such as MAWVWTLLFLMAAAQSIQA (SEQ ID NO: 103). Exemplarynucleotide sequences encoding the heavy chain of candidates 14EVQ and15EVQ with a leader sequence and the mature form (without a leadersequence) are shown in SEQ ID NOs: 104 and 105, respectively. Likewise,for expression, the light chain sequences of the antibodies of theinvention can include an N-terminal leader sequence such asMGVPTQVLGLLLLWLTDARC (SEQ ID NO: 106). Exemplary nucleotide sequencesencoding the light chain of codon optimized candidate 15 with a leadersequence and the mature form (without a leader sequence) are shown inSEQ ID NOs: 107 and 108, respectively.

Example 4 Characterization of Anti-TLR3 mAb Binding

EC50 values for the binding of the mAbs to human TLR3 extracellulardomain (ECD) were determined by ELISA. Human TLR3 ECD protein wasdiluted to 2 μg/ml in PBS and 100 μl aliquots were dispensed to eachwell of a 96-well plate (Corning Inc., Acton, Mass.). After overnightincubation at 4° C., the plate was washed 3 times in wash bufferconsisting of 0.05% Tween-20 (Sigma-Aldrich) in PBS. The wells wereblocked with 200 μl blocking solution consisting of 2% I-Block (AppliedBiosystems, Foster City, Calif.) and 0.05% Tween-20 in PBS. Afterblocking for 2 hours at room temperature the plate was washed 3 timesfollowed by addition of serial

TABLE 4 Candidate no. EC50 (ng/ml) 1 17.18 2 53.12 3 23.42 4 12.77 519.94 6 19 7 16.13 8 18.58 9 22.61 10 15.84 11 26.33 12 25.59 13 23.5114 33.59 15 32.64 16 43.66 17 13.8 18 9.68 19 66.54dilutions of the anti-TLR3 mAb candidates 1 to 19 in blocking buffer.The anti-TLR3 mAbs were incubated for 2 hours at room temperature andwashed 3 times. This was followed by addition of a peroxidase-conjugatedsheep anti-human IgG (GE Healthcare, Piscataway, N.J.) diluted 1:4000 inblocking buffer, incubated for 1 hour at room temperature followed by 3washes in wash buffer. Binding was detected by 10-15 minute incubationin TMB-S (Fitzgerald Industries International, Inc., Concord, Mass.).The reaction was stoppedwith 25 μl 2N H₂SO₄ and absorbance read at 450 nm with subtraction at650 nm using a SPECTRA Max spectrophotometer (Molecular Devices Corp.,Sunnyvale, Calif.). EC50 values were determined by non-linear regressionusing GraphPad Prism software (GraphPad Software, Inc., San Diego,Calif.).

EC50 values were determined for binding to huTLR3 (Table 4) byincubating with 100 μl of 4-fold serial dilutions of mAbs from 2.5 μg/mlto 0.6 μg/ml. An anti-human tissue factor mAb 859 and hu IgG4κ wereincluded as negative controls.

Binding affinity for huTLR3 ECD was also determined by Biacore analysis.The data (not shown) indicated that the mAbs 1-19 had a Kd for huTLR3ECD of less than 10⁻⁸ M.

Example 5 Competitive Epitope Binding

Epitope binding experiments were performed to determine the anti-TLR3antibody competition groups or “epitope bins”.

For competitive ELISA, 5 μl (20 μg/ml) of purified human TLR3 ECDprotein generated as described in Example 1 was coated on MSD HighBindplate (Meso Scale Discovery, Gaithersburg, Md.) per well for 2 hr atroom temperature. 150 μl of 5% MSD Blocker A buffer (Meso ScaleDiscovery) was added to each well and incubated for 2 hr at roomtemperature. Plates were washed three times with 0.1 M HEPES buffer, pH7.4, followed by the addition of the mixture of labeled anti-TLR3 mAbwith different competitors. Labeled antibodies (10 nM) were incubatedwith increasing concentrations (1 nM to 2 μM) of unlabeled anti-TLR3antibodies, and then added to the designated wells in a volume of 25 μlmixture. After 2-hour incubation with gentle shaking at roomtemperature, plates were washed 3 times with 0.1 M HEPES buffer (pH7.4). MSD Read Buffer T was diluted with distilled water (4-fold) anddispensed at a volume of 150 μl/well and analyzed with a SECTOR Imager6000. Antibodies were labeled with MSD Sulfo-Tag™ NHS-ester according tomanufacturer's instructions (Meso Scale Discovery).

The following anti-TLR3 antibodies were evaluated: mAbs 1-19 obtainedfrom a MorphoSys Human Combinatorial Antibody Library (shown in Table3a); c1068 (described in WO06/060513A2), c1811 (rat anti-mouse TLR3 mAbproduced by a hybridoma generated from rats immunized with mouse TLR3protein), TLR3.7 (eBiosciences, San Diego, Calif., cat no 14-9039) andIMG-315A (generated against human TLR3 amino acids amino acids 55-70(VLNLTHNQLRRLPAAN) from Imgenex, San Diego, Calif.). For mAbs 9, 10, 12,14 and 15, variants 9QVQ/QSV, 10QVQ/QSV, 12QVQ/QSV, 14EVQ or 15EVQ wereused in this study.

Based on competition assays, anti-TLR3 antibodies were assigned to fivedistinct bins. Bin A: mAbs 1, 2, 13, 14EVQ, 15EVQ, 16, 19; Bin B: mAbs3, 4, 5, 6, 7, 8, 9QVQ/QSV, 10QVQ/QSV, 11, 12QVQ/QSV, 17, 18; Bin C:antibody Imgenex IMG-315A; Bin D: antibodies TLR3.7, c1068; and Bin E:antibody c1811.

Example 6 Epitope Mapping

Representative antibodies from distinct epitope bins as described inExample 5 were selected for further epitope mapping. Epitope mapping wasperformed using various approaches, including TLR3 segment swappingexperiments, mutagenesis, H/D exchange and in silico protein-proteindocking (The Epitope Mapping Protocols, Methods in Molecular Biology,Volume 6, Glen E. Morris ed., 1996).

TLR3 Segment Swapping.

TLR3 human-mouse chimeric proteins were used to locate gross antibodybinding domains on TLR3. The human TLR3 protein extracellular domain wasdivided into three segments (aa 1-209, aa 210-436, aa 437-708 accordingto amino acid numbering based on human TLR3 amino acid sequence, GenBankAcc. No. NP_003256). MT5420 chimeric protein was generated by replacinghuman TLR3 amino acids 210-436 and 437-708 by corresponding mouse aminoacids (mouse TLR3, GenBank Acc. No. NP_569054, amino acids 211-437 and438-709). The MT6251 chimera was generated by replacing human aminoacids at positions 437-708 by mouse TLR3 amino acids (mouse TLR3,GenBank Acc. No. NP_569054, amino acids 438-709). All constructs weregenerated in the pCEP4 vector (Life Technologies, Carslbad, Calif.)using standard cloning procedures. The proteins were transientlyexpressed in HEK293 cells as V5-His6 C-terminal fusion proteins, andpurified as described in Example 1.

mAb c1068.

mAb c1068 bound human TLR3 ECD with high affinity but did not bind wellto murine TLR3. c1068 lost its ability to bind to both MT5420 andMT6251, demonstrating that the binding site was located within the aminoacids 437-708 of the WT human TLR3 protein.

mAb 12QVQ/QSV.

mAb 12QVQ/QSV bound both chimeras, indicating that the binding site formAb 12QVQ/QSV was located within the amino acids 1-209 of the human TLR3protein having a sequence shown in SEQ ID NO:2.

In Silico Protein-Protein Docking.

The crystal structure of mAb 15EVQ (see below) and the published humanTLR3 structure (Bell et al., J. Endotoxin Res. 12:375-378, 2006) wereenergy minimized in CHARMm (Brooks et al., J. Computat. Chem. 4:187-217,1983) for use as the starting models for docking. Protein docking wascarried out with ZDOCKpro 1.0 (Accelrys, San Diego, Calif.), which isequivalent to ZDOCK 2.1 (Chen and Weng, Proteins 51: 397-408, 2003) withan angular grid of 6 degrees. Known N-linked glycosylation site Asnresidues in human TLR3 (Asn 52, 70, 196, 252, 265, 275, 291, 398, 413,507 and 636) (Sun et al., J. Biol. Chem. 281:11144-11151, 2006) wereblocked from participating in the antibody-antigen complex interface byan energy term in the ZDOCK algorithm. 2000 initial poses were outputand clustered and the docking poses were refined and rescored in RDOCK(Li et al., Proteins 53:693-707, 2003). The 200 poses with the highestinitial ZDOCK scores and 200 top RDOCK poses were visually inspected.

Crystallization of Fab 15EVQ was carried out by the vapor-diffusionmethod at 20° C. (Benvenuti and Mangani, Nature Protocols 2:1633-51,2007). The initial screening was set up using a Hydra robot in 96-wellplates. The experiments were composed of droplets of 0.5 μl of proteinsolution mixed with 0.5 μl of reservoir solution. The droplets wereequilibrated against 90 μl of reservoir solution. The Fab solution in 20mM Tris buffer, pH 7.4, containing 50 mM NaCl was concentrated to 14.3mg/ml using Amicon Ultra-5 kDa cells. The screening was performed withthe Wizard I & II (Emerald BioSystems, Bainbridge Island, Wash.) andin-house crystallization screens. Fab 12QVQ/QSV was crystallized in asimilar manner.

X-ray diffraction data were collected and processed using the RigakuMicroMax™-007HF microfocus X-ray generator equipped with an Osmic™VariMax™ confocal optics, Saturn 944 CCD detector, and an X-Stream™ 2000cryocooling system (Rigaku, Woodlands, Tex.). Diffraction intensitieswere detected over a 270° crystal rotation with the exposure time of 120s per half-degree image. The X-ray data were processed with the programD*TREK (Rigaku). The structure was determined by the molecularreplacement method using the program Phaser or CNX (Accelrys, San Diego,Calif.). Atomic positions and temperature factors were refined withREFMAC using all data in the resolution range 15-2.2 Å for Fab 15EVQ and50-1.9 Å for Fab 12QVQ/QSV. Water molecules were added at the(F_(o)-F_(c)) electron density peaks using the cut-off level of 3σ. Allcrystallographic calculations were performed with the CCP4 suite ofprograms (Collaborative Computational Project, Number 4. 1994. The CCP4suite: programs for protein crystallography. Acta Crystallogr.D50:760-763). Model adjustments were carried out using the program COOT(Emsley et al., Acta Crystallogr. D60:2126-2132, 2004).

The resolved crystal structure of mAb 15EVQ showed that the antibodycombining site was characterized by a number of negatively chargedresidues in the heavy chain (D52, D55, E99, D106 and D109). Thus,recognition between mAb 15EVQ and TLR3 most likely involved positivelycharged residues. The protein-protein docking simulations performedsuggested that two large patches on TLR3 involving multiple positivecharge residues showed good complementarity to the antibody. Theresidues on TLR3 in the interface of the TLR3—anti-TLR3 antibodysimulated complexes were R64, K182, K416, K467, Y468, R488, R489 andK493.

Mutagenesis Studies.

Single and combination point mutations were introduced into surfaceresidues of TLR3 ECD in the regions identified above to contain theepitopes of mAb 12 and mAb 15EVQ and the mutant proteins were tested forantibody binding.

The nucleotide sequence encoding human TLR3 amino acids 1-703 (the ECD),(SEQ ID NO: 4; GenBank accession number NP_003256), was cloned usingstandard protocols. All mutants were generated by site directedmutagenesis using the Strategene Quickchange II XL kit (Stratagene, SanDiego, Calif.) according to the manufacturer's protocol, using theoligonucleotides shown in Table 5a. Mutations were verified by DNAsequencing. The proteins were expressed under a CMV promoter asC-terminal His-tag fusions in HEK293 cells, and purified as described inExample 1.

Binding Assays.

The binding activity of mAb 12QVQ/QSV and mAb 15EVQ to human TLR3 andgenerated variants was evaluated by ELISA. To expedite the process,mutants in the predicted mAb 15EVQ binding site were co-expressed in HEKcells by co-transfection of TLR3 ECD mutant containing a C-terminal Histag with mAb 12QVQ/QSV, followed by purification by metal affinitychromatography. The recovered sample was a complex of the TLR3 mutantwith mAb 12. This approach was feasible because the mAb 12QVQ/QSV andmAb 15EVQ binding sites are distant from one another; and thus, pointmutations at one site are unlikely to affect the epitope at the othersite. These complexes were used in the ELISA binding assays. 5 μl perwell of 20 μg/ml wild type TLR3 ECD or mutant proteins in PBS werecoated on an MSD HighBind plate (Meso Scale Discovery, Gaithersburg,Md.). The plates were incubated at room temperature for 60 min andblocked.

TABLE 5a Sequences of the sense oligonucleiotides are shown.The anti-sense oligonucleotides with complementary sequences wereused in the mutagenesis reaction. Seg ID Variant Oligo NO: R64E 5′CCTTACCCATAATCAACTCGAGAGATTACCAGCCGCCAAC 3′ 136 K182E 5′CAAGAGCTTCTATTATCAAACAATGAGATTCAAGCGCTAAAAAGTGAAG 3′ 137 K416E 5′CCTTACACATACTCAACCTAACCGAGAATAAAATCTCAAAAATAG 3′ 138 K467E/Y468A 5′GAAATCTATCTTTCCTACAACGAGGCCCTGCAGCTGACTAGGAACTC 3′ 139 R488/R489/K493E5′ GCCTTCAACGACTGATGCTCGAGGAGGTGGCCCTTGAGAATGTGGATAGCTCTCCTTC 3′ 140T472S/R473T/N474S 5′ GTACCTGCAGCTGTCTACGAGCTCCTTTGCCTTGGTCCC 3′ 141N196A 5′ GAAGAACTGGATATCTTTGCCGCTTCATCTTTAAAAAAATTAGAGTTG 3′ 169 Q167A5′ GTCATCTACAAAATTAGGAACTGCGGTTCAGCTGGAAAATCTCC 3′ 170 K163E 5′CTCATAATGGCTTGTCATCTACAGAATTAGGAACTCAGGTTCAGC 3′ 171 K147E 5′GAAAATTAAAAATAATCCCTTTGTCAAGCAGGAGAATTTAATCACATTAGATCTGTC 3′ 172 K145E5′ GAAAATTAAAAATAATCCCTTTGTCGAGCAGAAGAATTTAATCACATTAG 3′ 173 V144A 5′CAGAAAATTAAAAATAATCCCTTTGCAAAGCAGAAGAATTTAATCACATTAG 3′ 174 N140A 5′CCAACTCAATCCAGAAAATTAAAGCTAATCCCTTTGTCAAGCAGAAG 3′ 175 D116R 5′CAATGAGCTATCTCAACTTTCTCGTAAAACCTTTGCCTTCTGCAC 3′ 176 D536K 5′GTCTTGAGAAACTAGAAATTCTCAAGTTGCAGCATAACAACTTAGCAC 3′ 177 D536A 5′CTTGAGAAACTAGAAATTCTCGCATTGCAGCATAACAACTTAGCAC 3′ 178 K619E 5′CTAAAGTCATTGAACCTTCAGGAGAATCTCATAACATCCGTTG 3′ 179 K619A 5′CTCTAAAGTCATTGAACCTTCAGGCGAATCTCATAACATCCGTTGAG 3′ 180 E570R 5′CCACATCCTTAACTTGAGGTCCAACGGCTTTGACGAG 3′ 181 N541A 5′GAAATTCTCGATTTGCAGCATAACGCCTTAGCACGGCTCTGGAAAC 3′ 182 Q538A 5′GAGAAACTAGAAATTCTCGATTTGGCGCATAACAACTTAGCACGGC 3′ 183 H539E 5′CTAGAAATTCTCGATTTGCAGGAAAACAACTTAGCACGGCTCTG 3′ 184 H539A 5′CTAGAAATTCTCGATTTGCAGGCTAACAACTTAGCACGGCTCTG 3′ 185 N517A 5′CATTCTGGATCTAAGCAACAACGCCATAGCCAACATAAATGATGAC 3′ 186 Y465A 5′GAAAATATTTTCGAAATCTATCTTTCCGCCAACAAGTACCTGCAGCTGAC 3′ 187 R488E 5′GCCTTCAACGACTGATGCTCGAAAGGGTGGCCCTTAAAAATG 3′ 188 R489E 5′CTTCAACGACTGATGCTCCGAGAGGTGGCCCTTAAAAATGTGG 3′ 189 K467E 5′CGAAATCTATCTTTCCTACAACGAGTACCTGCAGCTGACTAG 3′ 190overnight in MSD Blocker A buffer (Meso Scale Discovery, Gaithersburg,Md.) at 4° C. The following day the plates were washed and theMSDSulfo-tag labeled mAb 15EVQ added at concentrations from 500 pM to 1pM for 1.5 hours. After washes the labeled antibody was detected usingMSD Read Buffer T and the plates were read using a SECTOR Imager 6000.To evaluate the binding activity of mAb 12QVQ/QSV to human TLR3 andvariants, co-expression was carried out with mAb 15EVQ and bindingELISAs were performed as described for mAb 15EVQ, except that thedetecting antibody was labeled mAb 12QVQ/QSV.

mAb 12QVQ/QSV:

The binding site for mAb 12QVQ/QSV was located within the amino acids1-209 of the human TLR3 protein as determined in the segment swapstudies. The following TLR3 mutants were evaluated: D116R, N196A, N140A,V144A, K145E, K147E, K163E, and Q167A. The wild type TLR3 and V144Amutant showed comparable binding to mAb 12QVQ/QSV (FIG. 6A). Theantibody did not bind to TLR3 D116R mutant and had significantly reducedbinding affinity to the K145E mutant. Thus, residues D116 and K145 whichare closely apposed on the surface of TLR3 were identified as keyepitope sites for mAb 12QVQ/QSV (FIG. 7A).

The two critical residues of the mAb 12QVQ/QSV binding epitope werelocated near the face of the dsRNA binding site at the N-terminalsegment of the TLR3 ectodomain (Pirher, et al., Nature Struct. & Mol.Biol., 15:761-763, 2008). The complete epitope will contain otherresidues in the neighboring regions, which were not revealed bymutational analyses performed. Not wishing to be bound to any particulartheory, it is believed that binding of mAb 12QVQ/QSV on its TLR3 epitopemay directly or indirectly interfere with dsRNA binding on TLR3ectodomain, thereby disrupting receptor dimerization and activation ofdownstream signaling pathways.

mAb 15EVQ:

The following TLR3 mutants were evaluated: R64E, K182E, K416E, Y465A,K467E, R488E, R489E, N517A, D536A, D536K, Q538A, H539A, H539E, N541A,E570R, K619A, K619E, a double mutant K467E/Y468A, a triple mutantT472S/R473T/N474S, and a triple mutant R488E/R489E/K493E. The wild typeTLR3, the R64E, K182E, K416E mutants and the triple mutantT472S/R473T/N474S showed comparable binding to mAb 15EVQ (FIG. 6B andTable 5b). The antibody did not bind to TLR3 mutants K467E, R489E,K467E/Y468A and R488E/R489E/K493E (FIGS. 6B and 6C). The remainingvariants showed intermediate binding with the R488E having the greatesteffect. All of these mutants bound to mAb 12QVQ/QSV. These resultsshowed that resides K467 and R489 were critical determinants of the mAb15EVQ epitope. Residue R488 also contributed to the epitope. Theseresidues were closely apposed on the same surface of TLR3 (FIG. 7A). Theresults also showed that residues Y465, Y468, N517, D536, Q538, H539,N541, E570, and K619, all on the same surface as K467, R488 and R489,contributed to the epitope. This conclusion was further supported by theH/D exchange studies with mAb 15EVQ. FIG. 7A shows binding epitope sitesfor mAbs 12QVQ/QSV and 15EVQ (black) and C1068 mAb (grey) superimposedon the structure of human TLR3. The epitope for mAb 15EVQ coversresidues Y465, K467, Y468, R488, R489, N517, D536, Q538, H539, N541,E570, and K619.

H/D Exchange Studies.

For H/D exchange, the procedures used to analyze the antibodyperturbation were similar to that described previously (Hamuro et al.,J. Biomol. Techniques 14:171-182, 2003; Horn et al., Biochemistry45:8488-8498, 2006) with some modifications. Recombinant TLR3 ECD(expressed from Sf9 cells with C-terminal His-tag and purified) wasincubated in a deuterated water solution for predetermined timesresulting in deuterium incorporation at exchangeable hydrogen atoms. Thedeuterated TLR3 ECD was captured on a column containing immobilized mAb15EVQ and then washed with aqueous buffer. The back-exchanged TLR3 ECDprotein was eluted from the column and localization of deuteriumcontaining fragments was determined by protease digestion and mass specanalysis. As a reference control, TLR3 ECD sample was processedsimilarly except it was exposed to deuterated water only after captureon the antibody column and then washed and eluted in the same manner asthe experimental sample. Regions bound to the antibody were inferred tobe those sites relatively protected from exchange and thus contain ahigher fraction of deuterium than the reference TLR3 ECD sample. About80% of the protein could be mapped to specific peptides. Maps of H/Dexchange perturbation of TLR3 ECD by mAb 15EVQ are shown in FIG. 7B.Only the segment of TLR3 around the portion affected by mAb 15EVQ isshown for clarity. The remainder of the protein extending to the aminoand carboxyl termini of TLR3 ECD was not affected appreciably.

The H/D exchange studies identified peptide segments 465YNKYLQL₄₇₁,₅₁₄SNNNIANINDDML₅₂₆ and ₅₂₉LEKL₅₃₂ of SEQ ID NO: 2 as regions whereexchange on TLR3 was particularly altered by binding to mAb 15EVQ. Byits nature, H/D exchange is a linear mapping method and usually cannotdefine which residues within the peptide segment are most affected byantibody binding. However, the extensive overlap between the H/Dexchange and mutational results gives added confidence that the surfaceshown in FIG. 7A is the binding site for mAb 15EVQ. This binding sitewas in same linear amino acid sequence region as previously describedfor mAb c1068 (PCT Publ. no. WO06/060513A2) but it was found to belocated on a completely non-overlapping surface (FIG. 7A) in agreementwith the lack of cross-competition between these antibodies.

The mAb 15EVQ binding epitope was spatially proximal to the dsRNAbinding site at the C-terminal segment on TLR3 (Bell et al., Proc. Natl.Acad. Sci. (USA) 103: 8792-8797, 2006; Ranjith-Kumar et al., J BiolChem, 282: 7668-7678, 2007; Liu et al., Science, 320: 379-381, 2008).Not wishing to be bound to any particular theory, it is believed thatbinding of mAb 15EVQ on its TLR3 epitope causes steric clashes with aligand dsRNA molecule and/or the dimer partner, preventing ligandbinding and ligand-induced receptor dimerization.

TABLE 5b Variant mAb 15 Variant mAb 12 wt TLR3 ECD +++ wt TLR3 ECD +++R64E +++ D116R − K182E +++ N140A ++ K416E +++ V144A +++ Y465A ++ K145E +K467E − K147E ++ R488E + K163E ++ R489E − Q167A ++ N517A ++ N196A ++D536K ++ D536A ++ Q538A ++ H539E ++ H539A ++ N541A ++ E570R ++ K619E ++K619A ++ K467E/Y468A − R488/R489/K493E − T472S/R473T/N474S +++

Example 7 Generation of Variants with Enhanced Thermal Stability

Structure-based engineering was conducted to generate antibody variantswith increased thermal stability, with simultaneous efforts to maintainthe biological activity and minimize immunogenicity.

mAb 15EVQ was selected for engineering. To minimize immunogenicity, onlygermline mutations predicted to be beneficial based upon structuralconsiderations were pursued. The VL and VH sequences of mAb 15EVQ (SEQID NO: 41 and SEQ ID NO: 216, respectively) were aligned with the humangermline genes using BLAST searches. The closest germline sequencesidentified were GenBank Acc. No. AAC09093 and X59318 for VH and VL,respectively. The following differences were identified between thegermline VH, VL and those of the mAb 15EVQ VH and VL sequences: (VH)V34I, G35S, F50R, A61S, and Q67H; (VL) G30S, L31S, and A34N. Theidentified sequence differences were mapped onto the crystal structureof the mAb 15EVQ, and residues predicted to alter packing and interfaceinteractions were selected for engineering. Based upon the crystalstructure of the antibody (see Example 6), potential structuredestabilizing residues were identified. (1) A small enclosed cavity wasidentified in the core of VH near V34. This cavity was large enough toaccommodate a slightly larger sidechain such as Ile. (2) E99 of VH CDR3was buried at the VH/VL interface without a H-bonding network. Thenegatively charged carboxylate group of E99 was in a generallyhydrophobic environment with mostly van der Waals (vdw) contacts toneighboring residues. Burying a charge group is usually energeticallyunfavorable and thus has destabilizing effect. (3) F50 of VH is a VH/VLinterface residue. Its aromatic sidechain is bulky and thus may havenegative impact upon the pairing. H-bonding and vdw packing networks forthe Fv were calculated and visually inspected in Pymol(www://_pymol_org). Buried cavities in the VH and VL domains werecomputed by Caver (Petrek et al., BMC Bioinformatics, 7:316, 2006). Allmolecular graphics figures were prepared in Pymol. Mutations were madeto the expression vectors encoding Fab fragments or IgG4 full humanantibodies generated as described in Example 3 using standard cloningtechniques using Quick Change II XL Site Directed Mutagenesis Kit(Stratagene, San Diego, Calif.), Change-IT Multiple Mutation SiteDirected Mutagenesis Kit (USB Corporation, Cleveland, Ohio) or QuickChange II Site Directed Mutagenesis Kit (Stratagene, San Diego, Calif.).The reactions were performed according to each manufacturer'srecommendations. The obtained clones were sequenced for verification,and the resulting engineered variants were named mAbs 15-1-15-10according to their modified heavy or light chain. Each variant chain (Hor L) was expressed with the wild type mAb 15EVQ L or H chain to produceantibodies, except that the heavy chain for mAb 15-10 was from mAb 15-6.A listing of the SEQ ID NOs: for the CDRs, variable regions of light andheavy chains and full length heavy and light chains for mAb 15EVQ andits engineered variants is shown in Table 6. Table 7 shows primers forgeneration of each variant.

TABLE 6 SEQ ID NO: Candidate Heavy Light no: HCDR1 HCDR2 HCDR3 LCDR1LCDR2 LCDR3 LV HV IgG4 chain 15 111 112 84 109 110 113 41 216 220 15615-1 111 114 84 109 110 113 41 124 130 156 15-2 115 112 84 109 110 11341 125 131 156 15-3 116 112 84 109 110 113 41 126 132 156 15-4 111 11784 109 110 113 41 127 133 156 15-5 116 118 84 109 110 113 41 128 134 15615-6 116 112 119 109 110 113 41 129 135 156 15-7 111 112 84 120 110 113122 42 102 157 15-8 111 112 84 121 110 113 123 42 102 158 15-9 116 118119 109 110 113 41 159 160 156 15-10 116 112 119 109 110 226 225 129 135227

Binding of mAbs 15-1-15-9 to TLR3 was evaluated by ELISA immunoassay.Human TLR3 ECD (100 μl of 2 μg/ml TLR3-ECD) was bound to a blackMaxisorb plate (eBioscience) overnight at 4° C. The plates were washedand blocked, and diluted antibodies were aliquoted at 50 μl per well induplicate onto the wells. The plate was incubated at RT for 2 hoursshaking gently. Binding was detected using luminescence POD substrate(Roche Applied Science, Mannheim, Germany, Cat. No. 11 582 950 001) andgoat anti-human Fc:HRP (Jackson ImmunoResearch, West Grove, Pa., Cat.No. 109-035-098) and the plate was read in a SpectraMax plate reader(Molecular Devices, Sunnyvale, Calif.).

DSC experiments were performed on a MicroCal's Auto VP-capillary DSCsystem (MicroCal, LLC, Northampton, Mass.) in which temperaturedifferences between the reference and sample cells were continuouslymeasured, and calibrated to power units. Samples were heated from 10° C.to 95° C. at a heating rate of 60° C./hour. The pre-scan time was 15minutes and the filtering period was 10 seconds. The concentration usedin the DSC experiments was about 0.5 mg/ml. Analysis of the resultingthermograms was performed using MicroCal Origin 7 software (MicroCal,LLC).

TABLE 7 Candidate Seq ID no: Mutants Primers NO: 15-1 HC: F50RGCCTGGAGTGGATGGGCCGGATCGACCCCAGCG 142 CGCTGGGGTCGATCCGGCCCATCCACTCCAGGC143 15-2 HC: V34I AGAGGTAACTCCCGTTGCGG 144GCATCTGGCGCACCCAGCCGATCCAGTAGTTGGTGAAG 145 15-3 HC: V34I/G35SAGAGGTAACTCCCGTTGCGG 146 GCATCTGGCGCACCCAGCTGATCCAGTAGTTGGTGAAG 147 15-4HC: A61S/Q67H AGAGGTAACTCCCGTTGCGG 144CGCTGATGGTCACGTGGCCCTGGAAGCTAGGGCTGTAGTTGG 148 TGTAG 15-5 HC:CTTCACCAACTACTGGATCAGCTGGGTGCGCCAGATGC 149 F50R/V34I/CGCTGATGGTCACGTGGCCCTGGAAGCTAGGGCTGTAGTTGG 148 G35S/A61S/Q67H TGTAG 15-6HC: CGCCATGTACTACTGCGCCCGCCAGCTGTACCAGGGCTAC 150 V34I/G35S/E99QGTAGCCCTGGTACAGCTGGCGGGCGCAGTAGTACATGGCG 151 15-7 LC: G30S/L31SGCCAGCCAGAGCATCAGCAGCTACCTGGCCTGGTACCAGC 152GCTGGTACCAGGCCAGGTAGCTGCTGATGCTCTGGCTGGC 153 15-8 LC: A34NAGAGGTAACTCCCGTTGCGG 144 CGGGCTTCTGCTGGTACCAGTTCAGGTAGCTGCTGATGCTCTG 15415-9 HC: CGCCATGTACTACTGCGCCCGCCAGCTGTACCAGGGCTAC 150 F50R/V34I/G35S/GTAGCCCTGGTACAGCTGGCGGGCGCAGTAGTACATGGCG 151 A61S/Q67H/E99QCAGGGCAACACCCTGCCCTACACCTTCGGCCAG 228 15-10 LC: S95PCTGGCCGAAGGTGTAGGGCAGGGTGTTGCCCTG 229

The thermal stability (Tm) of the generated variants was measured by DSC(Table 8). Binding of the antibody variants to TLR3 was comparable tothat of the parental antibody.

TABLE 8 Summary of melting temperatures (T_(M)) of the variants andrationale for making them. Candidate no: Mutations Rationale TM (° C.)ΔTM (° C.) 15EVQ WT 64.7 0 15-1 HV F50R VH/VL interface 69.3 4.6 15-2 HVV34I VH core packing 66.9 2.2 15-3 HV V34I/G35S H-bonding, VH core 71.26.5 packing 15-4 HV A61S/Q67H VH/VL packing, VH 65.4 0.7 surface charge15-5 HV F50R/V34I/G35S/ VH/VL interface, H- 76.2 11.5 A61S/Q67H bonding,VH core 15-6 HV V34I/G34S/E99Q H-bonding, VH core 75 10.3 packing,removal of 15-7 LV G30S/L31S L-CDR1 surface polar 63.1 −1.6 residues15-8 LV A34N VL/VH interface 64 −0.7 15-9 HV F50R/V34I/G35S/ VH/VLinterface, H- 76 11.3 A61S/Q67H/E99Q bonding, VH core 15-10 LV S95PCanonical structure 76.6 11.9 stabilization

Example 8 Generation of a Surrogate Anti-TLR3 Antibody

A chimeric antagonistic rat/mouse anti-mouse TLR3 antibody, herein namedmAb 5429 was generated to evaluate effects of inhibiting TLR3 signalingin various in vivo models, as the humanized antibodies generated inExample 1 did not have sufficient specificity or antagonist activity formouse TLR3. The surrogate chimeric mAb 5429 as well as its parent ratanti-mouse TLR3 antibody c1811 inhibited mouse TLR3 signaling in vitro,and in vivo, and ameliorated pathogenic mechanisms in several diseasemodels in the mouse.

Data discussed below suggests a role for TLR3 in the induction andperpetuation of detrimental inflammation, and contribute to therationale for the therapeutic use of TLR3 antagonists and TLR3 antibodyantagonists, for example acute and chronic inflammatory conditionsincluding hypercytokinemia, asthma and airway inflammation, inflammatorybowel diseases and rheumatoid arthritis, viral infections, and type IIdiabetes.

Generation of the Surrogate mAb 5429

CD rats were immunized with recombinant murine TLR3 ectodomain (aminoacids 1-703 of seq ID NO: 162, GenBank Acc. No. NP_569054) generatedusing routine methods. Lymphocytes from two rats demonstrating antibodytiters specific to murine TLR3 were fused to FO myeloma cells. A panelof monoclonal antibodies reactive to murine TLR3 were identified andtested for in vitro antagonist activity in the murine luciferasereporter and murine embryonic fibroblast assays. The hybridoma lineC1811A was selected for further work. Functional variable region geneswere sequenced from mAb c1811 secreted by the hybridoma. Cloned heavychain and light chain variable region genes were then respectivelyinserted into plasmid expression vectors that provided coding sequencesfor generating a chimeric Rat/Balb C muIgG1/κ mAb designated as mAb 5429using routine methods. The antibodies were expressed as described inExample 3. The amino acid sequences of the mAb 5429 heavy and lightchain variable regions are shown in SEQ ID NO:164 and SEQ ID NO: 163,respectively, and the heavy and light chain full length sequences areshown in SEQ ID NO:166 and SEQ ID NO: 165, respectively. The heavy andlight chain full length sequences of mAb c1811 are shown in SEQ ID NO:168 and SEQ ID NO: 167, respectively.

Characterization of mAb 5429

mAb 5429 was characterized in a panel of in vitro assays for itsneutralizing ability on TLR3 signaling. The activity assays and resultsare described below.

Murine Luciferase Reporter Gene Assay

The murine TLR3 cDNA (SEQ ID NO: 161, GenBank Acc. No: NM 126166) wasamplified by PCR from murine spleen cDNA (BD Biosciences, Bedford,Mass.), and cloned into the pCEP4 vector (Life Technologies, Carslbad,Calif.) using standard methods. 200 μl HEK293T cells were plated in 96well white clear-bottom plates at a concentration of 4×10⁴ cells/well incomplete DMEM, and used the following day for transfections usingLipofectamine 2000 (Invitrogen Corp., Carslbad, Calif.) using 30 ngpNF-κB firefly luciferase (Stratagene, San Diego, Calif.) or 30 ng pISREfirefly luciferase (BD Biosciences, Bedford, Mass.), 5 ng phRL-TKcontrol Renilla luciferase (Promega Corp., Madison, Wis.) reporterplasmids, 1.5 ng pCEP4 encoding the full-length murine TLR3, and 13.5 ngempty pcDNA3.1 vector (Life Technologies, Carslbad, Calif.) to bring thetotal DNA amount to 50 ng/well. 24 hours post-transfection, the cellswere incubated for 30 minutes to 1 hour at 37° C. with the anti-murineTLR3 antibodies in fresh serum-free DMEM before the addition of 0.1 or 1μg/μl poly(I:C). The plates were harvested after 24 hours using theDual-Glo Luciferase Assay System (Promega, Madison, Wis.). The relativelight units were measured using a FLUOstar OPTIMA multi-detection readerwith OPTIMA software (BMG Labtech GmbH, Germany). Normalized values(luciferase ratios) were obtained by dividing the firefly relative lightunits (RLUs) by the Renilla RLUs. mAb 5429 as well as its parent mAbc1811 and mAb 15 (Table 3a) reduced poly(I:C)-induced NF-kB and ISREactivation in a dose-dependent fashion (FIGS. 8A and 8B), demonstratingtheir abilities to antagonize the activity of TLR3. IC50s measured inthe ISRE assay were 0.5, 22, and 0.7 μg/ml for mAb 5249, mAB 15 and mAbc1811, respectively.

Murine Embryonic Fibroblast (MEF) Assay

C57BL/6 MEF cells were obtained from Artis Optimus (Opti-MEF™C57BL/6-0001). The cells were plated in 96-well flat bottom plates (BDFalcon) at 20,000 cells/well in 200 μl MEF media (DMEM with glutamax,10% heat inactivated-FBS, 1×NEAA, and 10 μg/ml gentamycin). Allincubations were done at 37° C./5% CO₂. 24 hours after plating, mAb 5429or mAb c1811 were added into wells. The plates were incubated with themAbs for 1 hr, after which Poly(I:C) was added at 1 μg/ml in each well.The supernatants were collected after a 24-hour incubation. Cytokinelevels were determined using a bead kit (Invitrogen Corp., Carslbad,Calif.) to detect CXCL10/IP-10 following manufacturer's protocol. Theresults were graphed using GraphPad Prism Software. Both antibodiesreduced poly(I:C)-induced CXCL10/IP-10 levels in a dose-dependentmanner, demonstrating the abilities of these antibodies to antagonizeendogenous TLR3 and inhibit TLR3 signaling (FIG. 9).

Flow Cytometry—Surface Staining

C57BL/6 and TLR3 knockout (TLR3KO) (C57BL/6 background; female, 8-12weeks of age, Ace Animals, Inc.), 10 per group, were dosedintraperitoneally with 1 ml of 3% Thioglycollate medium (Sigma) and 96hrs later, the mice were euthanized and the peritoneum from each mousewas lavaged with 10 ml sterile PBS. Thioglycollate-elicited peritonealmacrophages were resuspended in PBS and cell viability was assessedusing Trypan Blue staining. Cells were pelleted by centrifugation andresuspended in 250 μl FACS Buffer (PBS—Ca²⁺-Mg²⁺, 1% heat-inactivatedFBS, 0.09% Sodium Azide) and were kept on wet ice. The CD16/32 reagent(eBioscience) was used at 10 μg/10⁶ cells for 10 minutes to block FcReceptors on the macrophages. The cells were distributed at 10⁶ cells in100 μl/well for surface staining. Alexa-Fluor 647 (MolecularProbes)-conjugated mAb c1811 and mAb 1679 (rat anti-mouse TLR3 antibodythat had no TLR3 specificity, and thus used as an isotype control) wereadded at 0.25 μg/10⁶ cells and incubated on ice in the dark for 30minutes. The cells were washed and resuspended in 250 μl of FACS Buffer.The viability stain, 7-AAD (BD Biosciences, Bedford, Mass.), was addedat 5 μl/well no more than 30 minutes before acquisition of samples onFACS Calibur to detect a dead cell population. Samples were collected bythe FACS Calibur using Cell Quest Pro Software. FCS Express was used toanalyze the collected data by forming histograms.

The binding of mAb c1811 to murine thioglycollate-elicited peritonealmacrophages from C57BL/6 and TLR3KO mice were evaluated by flowcytometry to determine binding specificity. mAb 5429 was not used inthis assay since the mouse Fc region of this chimeric antibody wasexpected to contribute to non-specific binding. mAb c1811 exhibited nobinding to TLR3KO macrophages, and increased binding to the cellsurfaces of C57BL/6 peritoneal macrophages, suggesting a specificity ofthe mAb for TLR3 (FIG. 10). mAb 5429, having the same binding regions asmAb c1811, is assumed to have the same binding specificity as mAb c1811.

Example 9 TLR3 Antibody Antagonists Protect from TLR3-Mediated SystemicInflammation Model

The Poly(I:C)-induced systemic cytokine/chemokine model was used as amodel of TLR3-mediated systemic inflammation. In this model, poly(I:C)(PIC) delivered intraperitoneally induced a systemic cytokine andchemokine response that was partially TLR3-mediated.

Female C57BL/6 mice (8-10 weeks old) or female TLR3KO mice (C57BL/6background; 8-10 weeks old, Ace Animals, Inc.) were given mAb 5429 at10, 20 or 50 mg/kg in 0.5 ml PBS, mAb c1811 at 2, 10 or 20 mg/kg in 0.5ml PBS or 0.5 ml PBS alone (vehicle control) subcutaneously. 24 hoursafter antibody dosing, mice were given 50 μg poly(I:C) (Amersham Cat.No. 26-4732 Lot no. IH0156) in 0.1 ml PBS intraperitoneally.Retro-orbital blood was collected 1 and 4 hours after the poly(I:C)challenge. Serum was prepared from whole blood and analyzed for cytokineand chemokine concentrations by Luminex.

Results

Poly(I:C) delivered intraperitoneally induced a systemic cytokine andchemokine response that was partially TLR3-mediated, as evidenced by thesignificantly reduced production of a panel of chemokines and cytokinesin the TLR3KO animals (Table 9A). The TLR3-dependent poly(I:C)-inducedmediators were IL-6, KC, CCL2/MCP-1 and TNF-α at 1 hr post-poly(I:C)challenge, and IL-1α, CCL5/RANTES and TNF-α at 4 hr post-poly(I:C)challenge. Both mAb c1811 and mAb 5429 significantly reduced levels ofthese TLR3-dependent mediators, demonstrating the ability of theantibodies to reduce TLR3 signaling in vivo (Table 9B). Values in Table9 are shown as mean cytokine or chemokine concentrations in μg/ml of sixanimals/group ±SEM. These data suggest that TLR3 antagonism can bebeneficial in reducing excess TLR3-mediated cytokine and chemokinelevels in conditions such as cytokine storm or lethal shock.

TABLE 9A C57BL/6 TLR3KO PIC − + − + mAb 5429 (mg/kg) − − − − mAb c1811(mg/kg) − − − − 1 h PIC challenge TNFα 6.005 ± 0.32 319.4 ± 34.1*  9.13± 4.41 43.80 ± 10.13** KC 129.3 ± 9.83  2357 ± 491.5* 152.0 ± 21.34432.3 ± 90.66** IL-6 40.91 ± 5.66  5317 ± 856.7* 120.1 ± 99.99  1214 ±294.9** MCP-1 84.67 ± 18.45 694.6 ± 127.8* 67.85 ± 34.16 249.9 ± 55.60**4 h PIC challenge IL-1α 28.21 ± 17.78 796.7 ± 45.0* 13.94 ± 13.84 408.5± 29.91** RANTES 20.87 ± 1.738  4511 ± 783.4* 36.01 ± 4.484 706.3 ±84.36** TNFα  0.10 ± 0 561.7 ± 81.84* 3.215 ± 3.115 305.8 ± 53.63** *p <0.001: One Way ANOVA to C57BL/6 PBS **p < 0.001 One Way ANOVA to C57BL/6PIC

TABLE 9B C57BL/6 PIC + + + + + + mAb 5429 (mg/kg) 50 20 10 — — — mAbc1811 (mg/kg) — — — 20 10 2 1 h PIC challenge TNF-α 29.33 ± 3.78***31.05 ± 1.59*** 59.55 ± 12.71*** 32.54 ± 3.89*** 42.22 ± 7.04*** 42.61 ±10.58*** KC 466.3 ± 92.35*** 440.3 ± 10.01*** 744.6 ± 103.1** 637.3 ±151.0*** 944.2 ± 130.9** 919.3 ± 231.2** IL-6 480.2 ± 62.88*** 375.9 ±46.14*** 705.2 ± 149.8*** 739.2 ± 113.3***  1047 ± 222***  1229 ±378.4*** MCP-1 168.5 ± 15.04** 321.6 ± 206.7 219.2 ± 70.58* 184.0 ±14.92** 278.3 ± 53.57 414.9 ± 97.17 4 h PIC challenge IL-1α 343.0 ±33.01*** 452.6 ± 94.86** 481.1 ± 121.0* 354.8 ± 45.43*** 351.7 ±68.85*** 352.4 ± 39.60*** RANTES  1381 ± 169.7***  2439 ± 308.7**  1601± 398.9***  1303 ± 168.0***  1365 ± 474.1***  2209 ± 402.5** TNF-α 100.1± 8.5*** 205.1 ± 41.85*** 226.1 ± 64.72*** 138.9 ± 26.0*** 121.6 ±38.85*** 223.8 ± 47.74*** ***p < 0.001, **p < 0.01, *p < 0.05: One WayANOVA statistics were compared to the C57BL/6 + PIC group

Example 10 TLR3 Antibody Antagonists Reduce Airway HyperresponsivenessModel

Airway hyperresponsiveness was induced by Poly(I:C).

Female C57BL/6 mice (12 weeks old) or female TLR3KO mice (C57BL/6background; 12 weeks old, Ace Animals, Inc.) were anesthetized withisoflurane and several doses (10-100 μg) of poly(I:C) in 50 μl sterilePBS were administered intranasally. Mice received three administrationsof poly(I:C) (or PBS) with a 24 hour rest period between eachadministration. 24 hours following the last poly(I:C) (or PBS)administration, lung function and airway hyperresponsiveness tomethacholine were measured using whole body plethysmography (BUXCOsystem). The mice were placed into the whole body plethysmograph chamberand allowed to acclimate for at least 5 minutes. Following baselinereadings, mice were exposed to increasing doses of nebulizedmethacholine (Sigma, St. Louis, Mo.). The nebulized methacholine wasadministered for 2 minutes, followed by a 5-minute data collectionperiod, followed by a 10-minute rest period before subsequentincreasing-dose methacholine challenges. The increased airflowresistance was measured as Enhanced Pause (Penh) and is represented asthe average Penh value over the 5-minute recording period (BUXCOsystem). Following lung function measurements, mice were euthanized andthe lungs were cannulated. Bronchoalveolar lavages (BAL) were performedby injecting 1 ml of PBS into the lungs and retrieving the effluent. Thelung tissues were removed and frozen. BAL fluids were centrifuged (1200rpm, 10 min.) and the cell-free supernatants were collected and storedat −80° C. until analysis. Cell pellets were resuspended in 200 μl PBSfor total and differential cell counts. The multiplex assay wasperformed following the manufacturer's protocol and the MultiplexImmunoassay Kit (Millipore, Billercia, Mass.).

Results

Previous observations demonstrated that the intranasal administration ofpoly(I:C) induced a TLR3-mediated impairment in lung function in micewith increased enhanced pause (PenH) measurement in whole bodyplethysmography (Buxco) at baseline and an increased responsiveness toaerosolized methacholine (an indicator of airway hyperesponsiveness)(PCT Publ. No. WO06/060513A2). This impairment in the lung function wasassociated with neutrophil recruitment into the lung, and increasedlevels of pro-inflammatory cytokines/chemokines in the lung. In thisstudy, the effect of mAb 1811 and mAb 5429 was evaluated inpoly(I:C)-induced impairment in lung function by administering eachantibody at 50 mg/kg subcutaneously prior to poly(I:C) challenge.

TLR3-mediated impairment of lung function was significantly reduced bytreatment of animals with TLR3 antibody antagonists prior to thepoly(I:C) challenge. TLR3-mediated increases in baseline PenH and airwaysensitivity to methacholine were prevented in the anti-TLR3antibody-treated animals (FIG. 11). Further, TLR3-mediated recruitmentof neutrophils into the mouse lung and generation of chemokines in theairways were reduced in the anti-TLR3 antibody-treated animals. Theneutrophil numbers (FIG. 12) and the CXCL10/IP-10 levels (FIG. 13) weremeasured from the collected bronchoalveolar lavage fluid (BALF). Thestudies were repeated at least three times with similar results. Datashown in FIGS. 11, 12 and 13 are from one representative study. Eachsymbol represents a data point from one mouse, and the horizontal barsshow group means. The study demonstrated that systemically-administeredTLR3 antibody antagonists reached the lung, reduced TLR3-mediatedimpairment of lung function, neutrophil infiltration into the airway,chemokine generation and respiratory tract inflammation in the usedmodel. Thus, TLR3 antagonists may be beneficial in the treatment orprevention of respiratory diseases characterized by airwayhyperresponsiveness, such as asthma, allergic rhinitis, chronicobstructive pulmonary disease (COPD), and cystic fibrosis.

Example 11 TLR3 Antibody Antagonists Protect from Inflammatory BowelDisease Model

The DSS colitis Model was used as a model of inflammatory bowel disease.

Female C57BL/6 mice (<8 weeks old) or female TLR3KO mice (C57BL/6background; <8 weeks old weighing between 16.5 g and 18 g, Ace Animals,Inc.) were fed gamma-irradiated food starting on day −1. DSS (Dextransulfate) (MP Biomedicals, Aurora, Ohio, Catalog no: 160110; 35-50 kDa;18-20% Sulfur, Lot no. 8247J) was diluted in autoclaved acidifieddrinking water to a final concentration of 5%. The DSS-water wasadministered for 5 days, after which it was replaced with plain water.Mice were allowed to drink water ad libitum throughout the study. Allwater bottles were weighed every day to record water consumption. Ondays 0, 2, and 4 mice were dosed intraperitoneally with 5 mg/kg (0.1 mgin 0.1 ml PBS) mAb 5429, mouse anti-TNF-α antibody, or PBS as a control.Mice were monitored daily throughout the study and were weighed on days0 through 4 and day 7. Mice were euthanized on days 2 and 7 of thestudy. Abdominal cavities were opened and the ascending colons cut wherethey join the cecum. Colons were collected and fixed in 10% neutralbuffered formalin. Colons were paraffin-embedded, sectioned and H&Estained (Qualtek Molecular Labs, Santa Barbara, Calif.). Colonichistopathological assessments were done in a blinded fashion by aveterinary pathologist as described below (PathoMetrix, San Jose,Calif.).

Histopathologic Evaluation

Two segments of large intestine, colon and rectum were evaluated andscored for the following changes: (i) single cell necrosis; (ii)epithelial ulceration; (iii) epithelial sloughing; (iv) cryptal abscess;(v) cell proliferation; (vi) cryptal cell proliferation; (vii)granulation tissue formation in the lamina propria; (viii) granulationtissue in the submucosa; (ix) submucosal inflammatory cell infiltrate,neutrophil predominant; and (x) submucosal edema.

A single, overall score of severity was given based on the followingstandards:

-   -   0—non-existent    -   1—mild, focal or occasionally found    -   2—mild, multifocal    -   3—moderate, frequently found but in limited areas    -   4—severe, frequently found in many areas or extensions of the        tissue submitted    -   5—very severe, extends to large portions of the tissue submitted

Results

Previous observations demonstrated that TLR3KO animals showedsignificantly reduced histopathology compared with wild type mice in amodel of inflammatory bowel disease induced by DSS ingestion (PCT Publ.No. WO06/60513A2), thus suggesting that TLR3 signaling plays a role inthe pathogenesis in this model. It has been reported that commensalbacterial RNA or mammalian RNA released from necrotic cells can act asendogenous ligands to stimulate TLR3 signaling (Kariko et al., Immunity23165-231175 2005; Kariko et al., J. Biol. Chem. 279:12542-12550 2004),and therefore TLR3 stimulation by endogenous ligands in the gut mayenhance and perpetuate inflammation in the DSS colitis model.

Disease severity was ameliorated in DSS-exposed animals upon treatmentwith anti-TLR3 antibodies, as assessed by compound histopathology scores(FIG. 14). FIG. 14 shows means, standard deviations and 95% confidenceintervals for disease severity scores as horizontal bars. Significantreduction in the scores were observed in the wild type DSS-exposedanimals treated with anti-TLR3 antibodies (p<0.05) when compared tountreated wild type animals. DSS-exposed TLR3KO animals were protectedfrom DSS-induced changes. DSS-exposed animals receiving anti-mouse TNF-αmAb demonstrated no improvement in histopathology in the DSS model.Therefore, the DSS model may be useful in evaluating therapeutics thatmay target the human patient population that is non-responsive toanti-TNF-α therapies, and neutralizing anti-TLR3 antibodies may have thepotential to provide benefit to patients with inflammatory bowel diseasewho do not respond to anti-TNF-α therapies.

Model

The T cell Transfer Model was used as a model of inflammatory boweldisease. In this model, gut inflammation was induced in SCID mice by thetransfer of a population of regulatory T cell-devoid naïve T cells fromimmune-competent mice, which attack antigen-presenting cells in the gutmucosa.

Naïve T-cells (CD4+CD45RB^(high) T cells) were injectedintraperitoneally into SCID recipients to induce chronic colitis. Micewere given either PBS (500 μl/mouse intraperitoneally; vehicle control),mAb 5429 (0.1 mg/mouse intraperitoneally), or anti-TNF-α antibody (0.05mg/mouse intraperitoneally; positive control) beginning 48 hoursfollowing transfer of T-cells and then twice weekly throughout the 8week study. At 8 weeks following T-cell transfer (or when mice lost >15%of their original body weight) animals were euthanized and colonsremoved. Colons were fixed, paraffin-embedded and H&E stained.Histopathology (cell infiltration, crypt abscesses, epithelial erosion,goblet cell loss, and bowel wall thickening) was assessed quantitativelyin a blinded fashion.

Results

Disease severity was ameliorated in animals that received T-celltransfer upon treatment with anti-TLR3 antibodies, as assessed bysignificant reduction in the histopathology sum of scores when comparedto the control animals (p<0.05)(FIG. 15A). For the sum of scores, cryptabscesses, ulceration, neutrophil influx, goblet cell loss, abnormalcrypts, lamina propria inflammation and transmural involvement wasassessed. Significant reduction was observed with crypt abscesses,ulceration and neutrophil influx (for all p<0.05) (FIG. 15B). Anti-TNF-αantibody was used as a positive control at doses known to provideoptimal benefit.

Studies using two well known models of inflammatory bowel diseases, theDSS and the T-cell transfer model, demonstrated that systemicallydelivered TLR3 antibody antagonists reached the gut mucosa and reducedgastrointestinal tract inflammation induced through two differentpathogenic mechanisms. Thus, TLR3 antagonists may be beneficial for thetreatment of inflammatory bowel diseases, includinganti-TNF-α-refractory cases, and other immune-mediated pathologies inthe gastrointestinal tract.

Example 12 TLR3 Antibody Antagonists Protect from Collagen-InducedArthritis Model

The collagen-induced arthritis (CIA) model was used as a model ofrheumatoid arthritis.

Male B10RIII mice (6-8 weeks old, Jackson Labs) were divided into groupsof 15 per group (arthritis groups) or 4 per group (control mice).Arthritis groups were anesthetized with Isoflurane and given injectionsof Type II collagen (Elastin Products) and Freund's complete adjuvantsupplemented with M. tuberculosis (Difco) on days 0 and 15. On day 12,mice with developing type II collagen arthritis were randomized by bodyweight into treatment groups and were dosed subcutaneously (SC) on days12, 17, and 22 (d12, d17, 2d2) with mAb 5429 (25 mg/kg), the negativecontrol antibody CVAM (a recombinant mAb of no known specificity in themouse) (5 mg/kg) or anti-TNF-α antibody (5 mg/kg, positive control). Inaddition, control groups of mice were treated with vehicle (PBS) ordexamethasone (0.5 mg/kg, Dex, reference compound) subcutaneously (SC)daily (QD) on days 12-25. Animals were observed daily from days 12through 26. Fore and Hind paws were evaluated by a clinical scoringsystem (shown below). Animals were euthanized on study day 26 andhistopathology was assessed in a blinded fashion (scoring systemdescribed below). Efficacy evaluation was based on animal body weights,and clinical arthritis scores. All animals survived to studytermination.

Clinical Scoring Criteria for Fore and Hind Paws

-   -   0—normal    -   1—hind or fore paw joint affected or minimal diffuse erythema        and swelling    -   2—hind or fore paw joints affected or mild diffuse erythema and        swelling    -   3—hind or fore paw joints affected or moderate diffuse erythema        and swelling    -   4—marked diffuse erythema and swelling, or =4 digit joints        affected)    -   5—severe diffuse erythema and severe swelling entire paw, unable        to flex digits)        Histopathologic Scoring Methods for Mouse Joints with Type II        Collagen Arthritis

When scoring paws or ankles from mice with lesions of type II collagenarthritis, severity of changes as well as number of individual jointsaffected were considered. When only 1-3 joints of the paws or ankles outof a possibility of numerous metacarpal/metatarsal/digit ortarsal/tibiotarsal joints were affected, an arbitrary assignment of amaximum score of 1, 2 or 3 for parameters below was given depending onseverity of changes. If more than 2 joints were involved, the criteriabelow were applied to the most severely affected/majority of joints.

Clinical data for paw scores were analyzed using AUC for days 1-15, and% inhibition from controls were calculated.

Inflammation

-   -   0—normal    -   1—minimal infiltration of inflammatory cells in synovium and        periarticular tissue of affected joints    -   2—mild infiltration, if paws, restricted to affected joints    -   3—moderate infiltration with moderate edema, if paws, restricted        to affected joints    -   4—marked infiltration affecting most areas with marked edema    -   5—severe diffuse infiltration with severe edema

Pannus

-   -   0—normal    -   1—minimal infiltration of pannus in cartilage and subchondral        bone    -   2—mild infiltration with marginal zone destruction of hard        tissue in affected joints    -   3—moderate infiltration with moderate hard tissue destruction in        affected joints    -   4—marked infiltration with marked destruction of joint        architecture, most joints    -   5—severe infiltration associated with total or near total        destruction of joint architecture, affects all joints

Cartilage Damage

-   -   0—normal    -   1—minimal to mild loss of toluidine blue staining with no        obvious chondrocyte loss or collagen disruption in affected        joints    -   2—mild loss of toluidine blue staining with focal mild        (superficial) chondrocyte loss and/or collagen disruption in        affected joints    -   3—moderate loss of toluidine blue staining with multifocal        moderate (depth to middle zone) chondrocyte loss and/or collagen        disruption in affected joints    -   4—marked loss of toluidine blue staining with multifocal marked        (depth to deep zone) chondrocyte loss and/or collagen disruption        in most joints    -   5—severe diffuse loss of toluidine blue staining with multifocal        severe (depth to tide mark) chondrocyte loss and/or collagen        disruption in all joints

Bone Resorption

-   -   0—normal    -   1—minimal with small areas of resorption, not readily apparent        on low magnification, rare osteoclasts in affected joints    -   2—mild with more numerous areas of, not readily apparent on low        magnification, osteoclasts more numerous in affected joints    -   3—moderate with obvious resorption of medullary trabecular and        cortical bone without full thickness defects in cortex, loss of        some medullary trabeculae, lesion apparent on low magnification,        osteoclasts more numerous in affected joints    -   4—marked with full thickness defects in cortical bone, often        with distortion of profile of remaining cortical surface, marked        loss of medullary bone, numerous osteoclasts, affects most        joints    -   5—severe with full thickness defects in cortical bone and        destruction of joint architecture of all joints

Results

Dexamethasone (Dex) and anti-mouse TNF-α antibody was used as a positivecontrol, PBS was used as vehicle control, and CVAM was used as anegative control antibody. All treatments were initiated on day 12 ofthe study, during the development of joint disease. Disease incidencefor vehicle-treated disease control animals was 100% by study day 22.Negative control groups treated with vehicle or CVAM antibody had thehighest clinical scores. Significantly reduced clinical scores wereobserved for the groups treated with Dex (p<0.05 for d18-d26), 5 mg/kganti-TNF-α antibody (p<0.05 for d18-26), or 25 mg/kg mAb 5429 (p<0.05for d18-d23 and d25-d26) (FIG. 16). Clinical arthritis scores expressedas area under the curve (AUC) were significantly reduced by treatmentwith 25 mg/kg mAb 5429 (43% reduction), 5 mg/kg anti-TNF-α antibody(52%), or Dex (69%) as compared to vehicle controls. FIG. 17 shows meansand standard deviations for AUC for each group.

Histopathological effects of the treatments were also assessed. Paw boneresorption was significantly decreased by treatment with 25 mg/kg mAb5429 (47% decrease) as compared to vehicle controls. Positive controlmice treated with 5 mg/kg anti-TNF-α antibody had significantlydecreased paw inflammation (33%), cartilage damage (38%), and summed pawscores (37%). Treatment with Dex significantly reduced all pawhistopathology parameters (73% reduction of summed scores).

These data demonstrate that TLR3 antibody antagonists improve clinicaland histopathological disease symptoms in the CIA model, and suggest theuse of TLR3 antagonists for treatment of rheumatoid arthritis.

Example 14 TLR3 Antibody Antagonists Protect from Acute Lethal ViralInfections Model

An influenza A virus challenge model was used as a model of acute lethalviral infection.

On Day −1, 4, 8, and 12, female C57BL/6 mice (12 weeks old) or femaleTLR3KO mice (C57BL/6 background; 12 weeks old, ACE Animals, inc., 15mice per group) were dosed subcutaneously 20 mg/kg mAb 5429, or PBSalone. On day 0, the mice were anesthetized by isoflurane and wereintranasally administered Influenza A/PR/8/34 virus (ATCC, Rockland,Md., Lot no. 218171), in 25 μl PBS (equivalent to 10^(5.55)CEID50).Animals were observed two times a day for changes in body weight andsurvival over the period of 14 days. A clinical scoring system was usedto evaluate the level of disease progression and subtle improvements inresponse to Influenza A virus treatment.

Clinical Scores

-   -   0—normal, alert and reactive, no visible signs of illness    -   1—ruffled coat, with or without slightly reduced ambulation    -   2—ruffled coat, hunched posture when walking, reluctant        ambulation, labored breathing    -   3—ruffled coat, labored breathing, ataxia, tremor    -   4—ruffled coat, inability to ambulate with gentle prodding,        unconsciousness, feels cold to the touch    -   5—found dead

Results

Survival, daily clinical scores, and changes in body weight wereevaluated in the study. Both influenza A infected WT mice administeredmAb 5429 (20 mg/kg) and influenza A infected TLR3KO not receiving mAb5429 demonstrated a statistically significant increase in survival(p<0.001 and p<0.01, respectively) when compared to C57BL/6 miceinoculated with the Influenza virus, indicating that antagonism ordeficiency of TLR3 can prevent influenza-induced mortality (FIG. 18).Clinical scores were significantly reduced in the group receiving 20mg/kg mAb 5429, as well as in the TLR3KO group (FIG. 19). The bodyweight of the mice was observed over a period of 14 days after influenzavirus administration. Body weight decreased steadily in C57BL/6 micedosed with Influenza A virus. However, both the C57BL/6 mice dosed with20 mg/kg mAb 5429 and the TLR3KO mice demonstrated significantly greaterbody weight relative to the WT C57BL/6 mice inoculated with Influenzavirus (FIG. 20). These results demonstrated that TLR3 antibodyantagonists reduced clinical symptoms and mortality in an acute lethalinfluenza viral infection model, and suggested that TLR3 antagonists mayprovide protection for humans in acute infectious states.

Example 15 TLR3 Antibody Antagonists Improve Hyperglycemia and ReducePlasma Insulin Model

The Diet-induced obesity (DIO) model was used as a model ofhyperglycemia and insulin resistance, and obesity.

C57BL/6 WT animals (about 3 weeks old, Jackson Labs) and TLR3KO animals(C57BL/6 background; about 3 weeks old, Ace Animals, Inc.) weremaintained on a high fat diet for 12 to 16 weeks. Both TLR3KO and WTC57BL/6 mice were fed either with normal chow or high-fat diet (PurinaTestDiet cat. no. 58126) consisting of 60.9% kcal fat and 20.8% kcalcarbohydrates. Mice were maintained on a 12:12-h light-dark cycle, withwater and food ad libitum. The weight of each mouse within each groupwas measured weekly. mAb 5429 was given intraperitoneally twice a weekfor the first week followed by once a week dosing for total of 7 weeks.Fasting retro-orbital blood serum samples were used for insulinmeasurements at the time points indicated. Glucose tolerance tests wereperformed by i.p administration of glucose at 1.0 mg/g body weight afterovernight fast at week 7. In addition, fasting insulin and glucoselevels were measured.

HOMA-IR was determined from the equation based on the levels of fastingglucose and insulin levels (12) using following equation:HOMA-IR=((fasting glucose (mmol/1)×fasting insulin (mU/l))/22.5 (Wallaceet al., Diabetes Care 27:1487-1495, 2004). Fasting blood glucose (BG)was determined using glucose oxidase assay. Fasting insulin levels weredetermined using the insulin rat/mouse ELISA kit (Crystal Chem, cat. No.90060).

Results

After 12-16 weeks on high fat diet, the WT DIO animals werehyperglycemic and hyperinsulinemic. Glucose tolerance was improved inthe WT DIO animals but not in the TLR3KO DIO animals upon treatment withmAb 5429. Significantly reduced blood glucose levels were observed inmAb 5429 treated animals post glucose challenge at 60, 90, 120, and 180min when compared to control (PBS only) (FIG. 21A). About 21% reductionin AUC was observed in the mAb 5429 treated WT DIO animals when comparedto the WT DIO mice not receiving the mAb. Fasting insulin levels werealso reduced in the WT DIO animals treated with mAb 5429 (FIG. 22).TLR3KO DIO animals showed no improvement in fasting insulin upon mAb5429 treatment. Homeostatic model assessment (HOMA) analysis indicatedimproved insulin sensitivity in the WT DIO animals treated with mAb5429, but not in the TLR3KO DIO animals. The HOMA-IR values were14.0±9.8, 8.7±4.9, 9.0±3.0 for WT DIO, 5 mg/kg of WT DIO mAb 5429, and20 mg/kg of WT DIO mAb 5429 animals, respectively. No effect wasobserved in TLR3KO DIO animals.

The study demonstrated that TLR3 antibody antagonists improved insulinresistance and reduced fasting glucose in the DIO model without weightloss, suggesting that TLR3 antagonists may be beneficial for thetreatment of hyperglycemia, insulin resistance, and type II diabetes.

Example 16 TLR3 Antibody Antagonists Protect from Bacteria andVirus-Induced Inflammatory Responses Reagents

Nontypeable Haemophilus influenza (NTHi) strains 35, isolated from aCOPD patient with bacterial exacerbations, was obtained from Dr. T. F.Murphy (Buffalo VA Medical Center, Buffalo, N.Y.). Human rhinovirus 16was obtained from the American Type Culture Collection (ATCC) withTCID(50)=2.8×10⁷/ml.

NTHi Stimulation Assays

NHBE cells (Lonza, Wakersville, Md.) were seeded in Microtest 96-welltissue culture plates (BD Biosciences, Bedford, Mass.) at 1×10⁵/well.NTHi grown on agar plates for 16-20 hr were resuspended in growth mediumat ˜2×10⁸ cfu/ml, treated with 100 μg/ml gentamycin for 30 min. andadded at ˜2×10⁷/well to 96-well plates containing NHBEs. After 3 hours,supernatants were removed and replaced with fresh growth medium with orwithout antibodies (0.08 to 50 μg/ml final concentration). Afteradditional 24 hr incubation, presence of cytokines and chemokines incell supernatants was assayed in triplicate with a Cytokine 25-plex ABbead kit, Human (including IL-1β, IL-1RA, IL-2, IL-2R, IL-4, IL-5, IL-6,IL-7, IL-8, IL-10, IL12p40p70, IL-13, IL-15, IL-17, INF-α, IFN-α, IFN-γ,GM-CSF, MIP-1α, MIP-1β, IP-10, MIG, Eotaxin, RANTES and MCP-1) (LifeTechnologies, Carslbad, Calif.) in the Luminex 100IS multiplexfluorescence analyzer and reader system (Luminex Corporation, Austin,Tex.).

Rhinovirus Stimulation Assays

NHBE cells were seeded in Microtest 96-well tissue culture plates (BDBiosciences, Bedford, Mass.) at 1×10⁵ cells/well. The next day,antibodies (0.08 to 50 μg/ml final concentration) were added to NHBE orBEAS-2B cells and incubated for 1 hr, followed by addition of 10 μl/wellof rhinovirus. After additional 24 hr incubation, presence of cytokinesand chemokines in cell supernatants was assayed by luminex assays asdescribed above.

Results

mAb 15EVQ inhibited NTHi induced IP-10/CXCL10 and RANTES/CCL5 productionin a dose-dependent manner, while the control antibody, human IgG4(Sigma, St. Louis, Mo.), showed no inhibitory effect on NTHi stimulation(FIG. 23A). mAb 15EVQ also inhibited rhinovirus induced CXCL10/IP-10 andCCL5/RANTES production (FIG. 23B).

Example 17 TLR3 Antibody Antagonists Suppress Inflammatory Responses inAstroctyes Methods

Normal human astrocytes from 2 donors (Lonza, Walkersville, Md.) wereplated in a 24 well plate at 75,000 cells/well and allowed to attachovernight. The next day, the astrocytes were treated with 200 ng/mlpoly(I:C) and/or 10 μg/ml mAb 18 for 24 hours. Cytokines were measuredby Luminex.

Results

Poly(I:C)-induced production of IL-6, IL-8, IL-12, IFN-α, IFN-γ,CXCL9/MIG, CCL3/MIP-1a, CCL4, CCL5/RANTES and CXCL10/IP-10 wereinhibited by mAb 18, as shown in Table 10.

TABLE 10 IL-6 IL-8 IL-12 IFN-α IFN-γ Donor 1 untreated 876.0 ± 36.8539.7 ± 32.6 16.6 ± 0.5 28.8 ± 1.5 12.3 ± 0.3 mAb 18 1011.9 ± 57.4 1401.9 ± 49.7  17.1 ± 0.5 31.6 ± 0.7 10.4 ± 0.2 Poly(I:C) ol* ol 30.3 ±1.5 47.1 ± 3.1 35.9 ± 1.0 Poly(I:C) + mAb 18 2225.0 ± 108.1 6104.4 ±259.9 16.8 + 0.9 30.5 ± 1.6 11.7 ± 0.6 Donor 2 untreated 729.1 ± 7.1 248.2 ± 4.7    14 ± 0.5 19.5 ± 1.8 10.5 ± 0.5 mAb 18 779.0 ± 9.8  1132.6± 30.6  14.3 ± 0.6 20.8 ± 1.9 10.5 ± 0.1 Poly(I:C) ol ol 25.5 ± 0.4 36.3± 1.9 30.8 ± 0.9 Poly(I:C) + mAb 18 3393.3 ± 107.5 8660.4 ± 354.3 16.2 ±0.3 24.7 ± 1.2 12.6 ± 0.3 CXCL9/MIG CCL3/MIP-1a CCL4 CCL5/RANTESCXCL10/IP-10 Donor 1 untreated 12.6 ± 0.7   21 ± 0.9 14.8 ± 0.7 bl** blmAb 18 14.8 ± 1.7 19.5 ± 1.5 14.8 ± 1.1 bl bl Poly(I:C) 78.3 ± 4.81569.3 ± 36.9  159.7 ± 12.7 788.2 ± 94.9 461.4 ± 10.3 Poly(I:C) + mAb 1818.5 ± 1.6 31.2 ± 1.9 13.2 ± 0.9 bl  6.9 ± 0.5 Donor 2 untreated  9.9 ±1.6 12.3 ± 1.7 11.3 ± 0.3 bl bl mAb 18  8.9 ± 0.7 13.2 ± 1.5 11.1 ± 0.7bl bl Poly(I:C)   62 ± 3.8 1552.9 ± 41.1  140.7 ± 4.8  546.8 ± 21.7533.2 ± 15   Poly(I:C) + mAb 18 18.3 ± 2.7 66.6 ± 3.8 12.1 ± 0.8 bl 29.1± 6.2 *ol: over detection level **bl: below detection level

Example 18 TLR3 Antibody Antagonists Suppress Inflammatory Responses inEndothelial Cells Methods

HUVEC cells (Lonza, Walkersville, Md.) were cultured in serum-containinggrowth medium recommended by Lonza. Cells were resuspended in serum-freemedia (Lonza, Walkersville, Md.), plated in 96-well plates at 3×10⁵cells/ml, and incubated at 37° C., 5% CO2 for 24 hrs. Poly(I:C) (GEHealthcare, Piscataway, N.J.) was added at increasing concentrations(1.5 to 100 μg/ml) and incubated for another 24 hours at 37° C. Forcytokine inhibition assays, mAb 15EVQ was added to the cells at variousconcentrations (0-50 μg/ml) and incubated for 30 min, after which 20μg/ml poly(I:C) was added for 24 hours. Cell supernatants were collectedand cytokine levels were measured using the human cytokine 30-plex kitand Luminex MAP technology (Invitrogen Corp., Carslbad, Calif.). Tomeasure sICAM-1 expression, the HUVEC cells were treated with 20 μg/mlpoly(I:C) and various concentrations of mAb 15EVQ (0.8-50 μg/ml). Thecell supernatants were analyzed for sICAM-1 expression by ELISA (R&Dsystems). Cell viability was measured using the CellTiterGlo kit(Promega, Madison, Wis.).

Results

HUVEC cells produced the following cytokines in response to poly(I:C):IL-1RA, IL-2, IL-2R, IL-6, IL-7, CXCL8/IL-8, IL-12 (p40/p70), IL-15,IL-17, INF-α, IFN-α, IFN-γ, GM-CSF, CCL3/MIP-1a, CCL4/MIP-1β,CXCL10/IP-10, CCL5/RANTES, CCL2/MCP-1, VEGF, G-CSF, FGF-basic, and HGF(Table 11). mAb 15EVQ dose-dependently reduced levels of all cytokinesinduced by poly(I:C) (Table 12). The ability of mAb 15EVQ to reducepoly(I:C)-induced production of INF-α, CCL2/MCP-1, CCL5/RANTES, andCXCL10/IP-10 suggested that inhibition of TLR3-mediated activities mayprotect against leukocyte and T cell infiltration that can lead toatherosclerosis. Also, inhibition of VEGF by mAb 15EVQ suggested apotential benefit of TLR3 blockade in pathologies mediated by VEGFincluding angiogenesis in a variety of cancers and ocular diseases suchas age-related macular degeneration.

INF-α and IFN-γ function in leukocyte recruitment and increase theexpression of adhesion molecules on the activated endothelium (Doukas etal., Am. J. Pathol. 145:137-47, 1994; Pober et al., Am. J. Pathol.133:426-33, 1988). CCL2/MCP-1, CCL5/RANTES, and CXCL10/IP-10 have beenimplicated in monocyte and T cell recruitment and contribute to thedevelopment of atherosclerosis (Lundgerg et al., Clin. Immunol. 2009).The generation of VEGF by endothelial cells has been linked to abnormaltissue growth or tumors in a variety of cancers during angiogenesis(Livengood et al., Cell. Immunol. 249:55-62, 2007).

TABLE 11 Poly(I:C) μg/ml IL-6 CXCL8/IL-8 CCL2/MCP-1 10 848.8 + 50.912876.0 + 2314.0  11813.4 + 1420.9  5 751.3 + 2.1  11363.7 + 108.2 11365.7 + 113.1  2.5 607.1 + 91.6 10961.5 + 2200.7  11607.3 + 2155.7 1.25  419.2 + 178.4 9631.5 + 3675.8 11690.9 + 3189.9  0.63 263.8 + 81.46231.9 + 1568.0 9075.6 + 1152.2 0.31  183.5 + 168.3 5257.9 + 1855.08106.8 + 1193.1 0.16 111.9 + 72.5 4057.6 + 1127.4 6619.8 + 1728.2 nopoly(I:C) 0.00 1286.6 + 300.8  1360.1 + 245.4  Poly(I:C) μg/ml IL-2RIL-15 IL-17 100    784.4 + 45.4 61.3 + 12.5 43.8 + 5.3 50    718.6 +56.8 61.3 + 12.5 47.6 + 0   25    735.7 + 23.4 56.7 + 18.9 58.3 + 4.912.5  650.5 + 29.8 38.8 + 6.5   39.8 + 10.9 6.25 643.4 + 39.9 34.2 + 0  32.1 + 0   3.13 681.8 + 24.3 38.8 + 6.5  43.8 + 5.3 1.56 578.6 + 10.529.4 + 6.7  36.1 + 5.6 no poly(I:C) 0.0 0.0 0.0 Poly(I:C) μg/ml IFNαCXCL10/IP-10 CCL4/MIP-1β 100 50.7 + 0   3803.1 + 185.5 234.5 + 19.7 5044.9 + 1.7 2235.9 + 184.6 291.6 + 41.8 25 46.1 + 0   2403.0 + 271.9278.7 + 4.7  12.5 41.2 + 3.5 2185.4 + 64.9  243.8 + 63.4 6.25 36.1 + 0  2100.0 + 288.1 201.9 + 46.2 3.13 40.0 + 1.8 3553.2 + 197.1 191.5 + 20.81.56 42.5 + 1.7 2064.3 + 242.1 165.3 + 16.3 no poly(I:C) 0.0 0.0 0.0Poly(I:C) μg/ml RANTES TNFα VEGF 100  6266.9 + 1708.7 12.8 + 3.2  581.1 + 181.4 50 2919.7 + 119.4 11.5 + 3.2  637.9 + 47.7 25 2805.1 +176.7 9.8 + 2.8 700.3 + 62.5 12.5 2598.6 + 68.6  7.3 + 0.9 513.2 + 73.56.25 2449.2 + 830.6 6.9 + 1.4 440.4 + 29.5 3.13 3117.1 + 795.7 7.3 + 0.9393.9 + 40.2 1.56 2481.0 + 719.3 6.0 + 1.8 358.4 + 74.8 no poly(I:C) 4.9 + 4.5 1.9 + 0.4 32.1 + 8.8 concentrations shown as pg/ml

Soluble Intercellular Adhesion Molecule 1 (sICAM-1) is generated byproteolytic cleavage and is a marker for endothelial cell activation.ICAM-1 plays a key role in leukocyte migration and activation and isupregulated on endothelial cells and epithelial cells duringinflammation where it mediates adhesion to leukocytes via integrinmolecules LFA-1 and Mac-1. Poly(I:C) activated the endothelial cells toupregulate sICAM-1 expression and the uregulation was reduced bytreatment with mAb 15EVQ (FIG. 24A).

TABLE 12 mAb 15 (μg/ml) 50.00 10.00 2.00 0.40 0.08 PIC + + + + + IL-6177.8 ± 5.6*  214.6 + 3.6*  359.2 + 57.6*   727.2 + 50.5* 10000 + 0  CXCL8/IL-8 1040.7 ± 185.9  1765.9 + 97.1   6460.3 + 3684.4  57349.5 +6293.4 72422.8 + 88279.2 CCL2/MCP-1 1187.7 ± 165.4*  1955.4 + 72.7* 9054.4 + 4110.9* 20000 + 0.0   20000 + 0.0   IL-2R 25.0 ± 35.3* 0.0 +0.0* 312.3 + 137.6* 521.5 + 5.5  664.7 + 9.8  IL-15 0.0 ± 0.0* 0.0 +0.0* 0.0 + 0.0*  4.1 + 0.0* 38.8 + 6.5  IL-17 1.3 ± 1.8* 11.8 + 16.8 11.8 + 16.8  27.9 + 6.0 47.4 + 10.4 IFNα 0.9 ± 1.3* 0.9 + 1.3* 19.0 +7.7*  36.1 + 0.0 44.9 + 1.7  CXCL10/IP-10 0.0 ± 0.0* 58.1 + 2.6* 633.0 + 471.6* 1441.4 + 97.1  3023.8 + 166.1  CCL4/MIP-1β 0.0 ± 0.0*0.0 + 0.0* 2.9 + 4.1*  62.1 + 7.2* 176.6 + 21.3* RANTES 3.0 ± 0.0*15.4 + 4.5*  201.1 + 169.1*  952.4 + 41.1* 2454.4 + 98.5*  TNFα 1.9 ±0.4* 1.6 + 0.0* 2.2 + 0.0*  3.4 + 0.0 6.3 + 0.5 VEGF 87.2 ± 8.7*  28.6 +8.7*  88.3 + 52.1* 156.1 + 6.4* 479.6 + 14.1  mAb 15 (μg/ml) 0.016 0.0030 PIC + + − IL-6 10000 + 0   10000 + 0   10000 + 0   CXCL8/IL-847047.5 + 52393.1 76066.5 + 11354.1  96478.0 + 122298.4 CCL2/MCP-120000 + 0.0   20000 + 0.0   20000 + 0.0   IL-2R 661.2 + 14.8  698.4 +57.6  654.2 + 14.8 IL-15 43.4 + 0.0  38.8 + 6.5  43.4 + 0.0 IL-17 54.3 +20.2 40.0 + 0.0  51.2 + 5.1 IFNα 41.2 + 3.5  47.3 + 1.7  40.0 + 1.8CXCL10/IP-10 2107.5 + 372.6  2346.4 + 226.1  2157.4 + 282.7 CCL4/MIP-1β227.5 + 19.9  248.3 + 19.4  281.7 + 37.5 RANTES 2698.1 + 88.6*  2624.4 +129.8* 3459.7 + 181.8 TNFα 8.5 + 0.0 7.6 + 1.4  6.9 + 2.3 VEGF 544.6 +8.3  533.5 + 70.2  607.3 + 29.9 *Indicates significant p-values (lessthan 0.05) comparing mAb15 concentration vs. poly(I:C) alone Values aremean (pg/ml) ± SEM

This suggested that TLR3 antibody antagonists can inhibit leukocytetrafficking and thus tissue damage caused by the influx of inflammatorycells.

For viability assays, HUVECs were cultured, plated and stimulated withpoly(I:C) as described above. mAb 15EVQ dose-dependently restoredpoly(I:C)-induced reduction in HUVEC cell viability (FIG. 24B).

Down-modulation of endothelial cell activation can suppress excessiveimmune cell infiltration and reduce tissue damage caused by cytokinesthat are increased during inflammatory conditions. Inflammation andoverexpression of cytokines and adhesion molecules on endothelial cellsare key contributors to developing atherosclerosis and hypertension.These data provide rationale for exploring the potential benefit of TLR3antagonists for use in diseases of the blood vessels includingvasculitides, and those featuring endothelial dysfunction. Anotherdisease that is affected by inflammation and overexpressed cytokines isKaposi's sarcoma (KS) that is common in immunosuppressed and HIVinfected individuals and is caused by Kaposi's sarcoma herpes virus(KSHV). VEGF and cytokine production contribute to the survival of KScells (Livengood et al., Cell Immunol. 249:55-62, 2007). TLR3antagonists could be beneficial at reducing angiogenic risks associatedwith KS and other tumors and at preventing cell viability loss andprotecting endothelial barrier integrity to prevent vascular leakage, apotentially serious condition associated with organ failure andlife-threatening inflammatory conditions such as sepsis. TLR3 antagonismmay also be beneficial in viral infections involving endothelial cellpathology such as the viral hemorrhagic fevers caused by members of thefamilies flaviviridae (e.g. Dengue, yellow fever), filoviridae (Ebola,Marburg), bunyaviridae (e.g. Hantavirus, Nairovirus, Phlebovirus), andarenaviridae (e.g. Lujo, Lassa, Argentine, Bolivian, Venezuelanhemorrhagic fevers (Sihibamiya et al., Blood 113:714-722, 2009).

Example 20 Cross-Reactivity of TLR3 Antibody Antagonists with Cynomolgusand Murine TLR3

Activity against cynomolgus or murine TLR3 were assessed using the ISREreporter gene assay as described in Example 2. The cynomolgus (SEQ IDNO: 217) and murine TLR3 cDNAs (SEQ ID NO: 161) were amplified fromwhole blood and cloned into the pCEP4 vector (Clontech), and expressedas described above. mAb 15EVQ had IC50s of 4.18 μg/ml and 1.74 μg/ml inthe cyno NF-κB and ISRE assays, respectively, compared to IC50s of 0.44and 0.65 μg/ml in the human TLR3 NF-kB and ISRE assays, respectively.Isotype control antibodies had no effect in these assays.

Example 21 Therapeutic Dosing of TLR3 Antibody Antagonists Protect fromAcute Lethal Viral Infections

Example 14 describes prophylactic treatment (dosed on days −1, 4, 8, and12) with TLR3 antibody antagonists against influenza A infection. Thisexample demonstrates that therapeutic dosing of TLR3 antibodyantagonists (day 3 after influenza A infection after the onset ofclinical symptoms) are efficacious in enhancing survival.

Model

An influenza A virus challenge model was used as a model of acute lethalviral infection as described in Example 14, except that dosing ofanimals with mAb 5249 was done 3 days post infection with influenza A,and the animals dosed were 8 weeks old. Anti-mouse IgG1 isotype controlmAb was from BioLegend. The animals were dosed days 3, 7 and 11post-infections with influenza A.

Survival, daily clinical scores, and changes in body weight wereevaluated in the study. Both the C57Bl/6 mice administered mAb 5249 andthe TLR3KO mice demonstrated a statistically significant increase insurvival (p<0.028 and p<0.001, respectively) relative to the C57BL/6mice inoculated with the anti-mouse IgG1 isotype control mAb andInfluenza virus (FIG. 25). The clinical scores were reduced (FIG. 26)and the body weights increased (FIG. 27) in the C57BL/6 mice dosed withmAb 5249 and in the TLR3KO animals when compared with C57BL/6 mice dosedwith anti-mouse IgG1 isotype control mAb and Influenza A. These resultsdemonstrated that TLR3 antibody antagonists reduced clinical symptomsand mortality in an acute lethal influenza viral infection model, andsuggested that TLR3 antagonists may provide protection for humans inacute infectious states.

Example 22 Epitopes and Paratopes of TLR3 Antibody Antagonists by X-RayCrystallography

The human TLR3 extracellular domain was crystallized in complex withFabs of mAb 15EVQ, mAb 12QVQ/QSV and mAb c1068.

Methods Expression and Purification of Proteins

The expression and purification of the TLR3 ECD (amino acids 1-703 ofSEQ ID NO: 2) the three Fabs were as described above.

Preparation of the TLR3 ECD-Three Fab Quaternary Complex

4 mg of human TLR3 ECD was mixed with 2.4 mg of each Fab and incubatedat 4° C. for 3.5 h, corresponding to a molar ratio of 1 TLR3 ECD:1.1Fab. The complex was purified by anion exchange chromatography on aMonoQ 5/50 GL column (GE Healthcare, Piscataway, N.J.), equilibratedwith 20 mM Tris pH 8.5, 10% glycerol (buffer A) and eluted with 20 mMTris pH 8.5, 10% glycerol, 1 M NaCl (buffer B). Approximately 2.48 mg ofcomplex in 1.74 mL was diluted to 10 mL with buffer A, loaded onto thecolumn at 1 mL/min and eluted with a linear gradient of 0-40% B over 40column volumes. Five consecutive purification runs were performed.Fractions from peak 1 were pooled, concentrated with an Amicon-15 mLUltra-30000 MWCO and a Microcon 30000 MWCO to 14.49 mg/mL in 20 mM TrispH 8.5, 27 mM NaCl, 10% glycerol (Extinction coefficient: A₂₈₀ (1mg/mL)=1.31).

Crystallization

Automated crystallization screening was performed using the Oryx4automatic protein crystallization robot (Douglas Instruments) dispensingequal volumes of protein and reservoir solution in a sitting drop formatusing Corning plate 3550 (Corning Inc., Acton, Mass.). Initial screeningwas with Hampton Crystal Screen HT (HR2-130, Hampton Research, AlisoViejo, Calif.). Small crystals from several conditions were used togenerate seeds, which were then used in Microseed-Matrix Screening(MMS). Several rounds of refinement were performed that were based onconditions from the initial screening that gave small crystals.Reservoir conditions used for MMS were based on those that gave smallcrystals after refinement: 18-28% polyethylene glycol (PEG) 3350, 1MLiCl, pH4.5 and 2.0-2.9 M (NH₄)₂SO₄, 5% PEG400, pH 4.5, and explored pHand different additives. MMS crystallization screening was performedusing the Oryx4 automatic protein crystallization robot (DouglasInstruments) by dispensing components in the following volume ratio: 1protein solution: 0.25 seed stock: 0.75 reservoir solution. Crystalsdiffracting to ˜10-Å resolution grew from 0.1 M Na acetate pH 4.5, 2.9 M(NH₄)₂SO₄, 5% methyl-pentane-diol (MPD) and 0.1 M Na acetate pH 4.5, 26%PEG3350, 1 M LiCl.

In an effort to improve the resolution of the crystals, MMS with theabove conditions was combined with additive screening using selectedcomponents of the Hampton Additive Screen HR2-428 (Hampton Research,Aliso Viejo, Calif.) in the following volume ratio: 1 protein solution:0.125 seed stock: 0.2 additive solution: 0.675 reservoir solution. X-rayquality crystals of the TLR3 ECD complexed with the Fabs, which diffractto ˜5-Å resolution, were obtained after applying a combination of MMSand Additive screening from a solution containing 0.1 M Na acetate pH4.5, 28% PEG 3350, 1 M LiCl, and 30 mM Gly-Gly-Gly.

X-Ray Data Collection of TLR3 ECD Quaternary Complex

For X-ray data collection, a crystal (size ˜1.0×0.5×0.1 mm³) was soakedfor a few seconds in a synthetic mother liquor (0.1 M Na acetate, pH4.5, 28% PEG 3350, 1 M LiCl, 16% glycerol), and flash frozen in thestream of nitrogen at 100 K. X-ray diffraction data were collected andprocessed using a Rigaku MicroMax™-007HF microfocus X-ray generatorequipped with an Osmic™ VariNax™ confocal optics, Saturn 944 CCDdetector, and an X-Stream™ 2000 cryocooling system (Rigaku, Woodlands,Tex.). Diffraction intensities were detected over a 250° crystalrotation with the exposure time of 1 min per half-degree image to themaximum resolution of 5 Å. The X-ray data were processed with theprogram D*TREK (Pflugrath, Acta Crystallographica Section D,55:1718-1725, 1999). The crystal belongs to the monoclinic space groupC2 with unit cell parameters: a=214.90 Å, b=142.08 Å, c=125.04 Å, andβ=103.17°. The asymmetric unit contains 1 molecule of the complex. TheX-ray data statistics are given in Table 13.

TABLE 13 Data Collection Space group C2 Unit cell axes (Å) 214.90,142.08, 125.04 Unit cell angles (°) 90, 103.17, 90 Resolution (Å) 30-5.0(5.18-5.00) No. unique reflections 15,877 (1589) Completeness (%) 99.8(99.6) Redundancy 5.2 (4.9) R_(merge) ^(a) 0.121 (0.312) <I/σ> 7.1 (2.9)Structure refinement Resolution (Å) 29.4-5.0 R_(cryst)/R_(free)(%)^(b)26.8/30.0 No. of reflections Working set 15,792 Test set (5% data) 788Rmsd from ideal values Bond length (Å) 0.007 Bond angels (°) 0.744Number of protein atoms 15,442 Ramachandran plot^(c) Favored regions (%)93.1 Allowed (%) 98.8 Disallowed (%) 1.2

Structure Determination

The crystal structure of the TLR3 ECD-ab 15EVQ-Fab 12QVQ/QSV-Fab c1068was determined by molecular replacement using Phaser (Read, ActaCrystallogr. D. Biol. Crystallogr.

57:1373-1382, 2001). The search models were TLR3 ECD (Protein DataBank(PDB) structure ID 1ziw with all glycans removed, Choe et al., Science309:581-585, 2005) and the high resolution crystal structures of thethree Fabs determined (See Table 13 for a summary of the crystal dataand refinement statistics for these Fab structures). The elbow angle ofFab 12QVQ/QSV was found to deviate significantly from that in the freeform. A series of Fab 12QVQ/QSV models were generated by adjusting theelbow angle at ˜5° intervals, one of which was found to agree well withthe electron density. The structure refinement was carried with PHENIX(Adams et al., J. Synchrotron Radiat. 11:53-55, 2004). The structure wasrefined as rigid body domains (each V or C domain) for the Fabs and 13rigid segments (Definitions used in the refinement: 30-60, 61-108,109-156, 157-206, 207-257, 258-307, 308-363, 364-415, 416-464, 465-514,515-570, 571-618, 619-687) for the TLR3 ECD with one B factor for eachFab rigid body and a single B for the entire TLR3 ECD.

Translation/Libration/Screw (TLS) refinement was introduced for each ofthe Fab rigid bodies and TLR3 ECD was divided into 2 TLS segments atresidue 330 of SEQ ID NO: 2. Glycan density was visible for 10 of the 15N-glycosylation sites. Carbohydrate models from the crystal structure ofthe human TLR3 extracellular domain (Choe et al., Science 309:581-585,2005, PDB structure ID: 1ziw) were then added. The density for a shortmissing segment in TLR3 ECD (residues 337-342 of SEQ ID NO: 2) wasvisible after rigid body refinement, and it was filled in with thecorresponding segment from the TLR3 extracellular structure 2a0z (Bellet al., Proc. Natl. Acad. Sci. (USA) 102:10976-10980, 2005, PDBstructure ID: 2a0z). The C-terminus of TLR3 ECD contained additionaldensity that matches that of 2a0z. This segment (647-703 of SEQ ID NO:2) was then replaced with (residues 647-687) of 2a0w. Thus, the TLR3 ECDmodel was a hybrid between the TLR3 structures 1ziw and 2a0z and refinedas 13 rigid body segments (amino acid range: 30-60, 61-108, 109-156,157-206, 207-257, 258-307, 308-363, 364-415, 416-464, 465-514, 515-570,571-618, 619-687).

The LCDR3 of Fab 12QVQ/QSV apparently adopted different conformationfrom its free form. Multi-start simulated annealing was carried out withstandard parameters in PHENIX. The models of this LCDR3 were visuallyinspected in the electron density map and the “best-matching”conformation was grafted onto the original model. The refinement processwas monitored by R_(free) against 5% of the reflections set aside priorto initiating the calculations. In the final round, one B factor foreach residue was included. Model inspection and manual rebuilding of theelbow regions of the Fabs and side chains at the protein-proteininterfaces were done using COOT (Emsley et al., Acta Crystallogr. D.Biol. Crystallogr. 60:2126-32, 2004). The final R_(cryst) and R_(free)were 26.8% and 30.0%, respectively, for all 15,792 independentreflections to 5.0 Å. The refinement statistics are given in Tables 13and 14.

Results The Molecular Structure of the TLR3 ECD-Three Fab QuaternaryComplex

The overall molecular structure of the complex is shown in FIGS. 28A and28B. In the asymmetric unit there is one TLR3 ECD and one molecule ofeach Fab. The structural model for TLR3 ECD includes all residues from30 to 687 of huTLR3 (SEQ ID NO: 2). For the three Fabs, all residuesfrom their respective unbound forms were included except solvent ionsand water molecules. The TLR3 ECD molecule is very similar to thepreviously reported structures in overall topology (rmsd of 0.79 Å for1ziw, 613 Cα's, and 1.37 Å for 2a0z, 595 Ca's). The Fab structures areall identical to their respective unbound forms except for LCDR3 of Fab12QVQ/QSV as described in Methods as well as the elbow regions and someside chains at TLR3 ECD/Fab interfaces.

TABLE 14 Data collection Fab 12QVQ/QSV Fab 15EVQ Fab c1068 Space groupP2₁ P2₁ P2₁ Cell dimensions a, b, c (Å) 75.83, 80.35, 83.06 54.68,74.74, 64.99 82.48, 136.94, 83.25 α, β, γ (°) 90, 115.24, 90 90, 103.69,90 90, 114.95, 90 Resolution (Å) 70-2.5 (2.59-2.50) 49-2.2 (2.28-2.20)50-1.9 (2.0-1.9) Unique reflections 27,785 (1653) 24,439 (1859) 117,490(5916) Completeness (%) 88.5 (53) 94.2 (72.8) 89.3 (45.2) Redundancy 4(1.8) 5.2 (4.3) 3.2 (2) R_(merge) ^(a) 0.164 (0.297) 0.088 (0.445) 0.065(0.264) <I/σ> (unaveraged) 2.9 (1.2) 3.8 (1.4) 5.7 (1.6) StructureRefinement Resolution (Å) 15-2.5 (2.56-2.50) 15-2.2 (2.26-2.20)75.38-1.90 (1.94-1.90) R_(cryst)/R_(free) (%)^(b) 19.7/25.4 (30.8/40.8)19.3/26.9 (24.6/31.1) 20.4/27.7 (39.8/51.1) No. of reflections Workingset 26,723 23,308 111,413 Test set 882 1,008 5,917 Number of atomsProteins 7,046 3,705 13,421 Solvent (water, etc.) 486 333 1,779 RMSDbond lengths (Å) 0.012 0.013 0.023 RMSD bond angles (°) 1.6 1.5 2Ramachandran plot^(c) Favored regions (%) 92.3 96.8 97.2 Allowed (%)98.9 99.3 99.7 Disallowed (%) 1.1 0.7 0.3 Values for highest resolutionshell are in ( )'s. ^(a)R_(merge) = Σ|I − <I>|/ΣI, where I is theintensity of the measured reflection and <I> is the mean intensity ofall measurements of this reflection. ^(b) ₌R_(cryst) = Σ||F_(obs)| −|F_(calc)||/Σ|F_(obs)|, where F_(obs) and F_(calc) are observed andcalculated structure factors and R_(free) is calculated for a set ofrandomly chosen 5% of reflections prior to refinement. ^(c)TheRamachandran plot was calculated with MolProbity (Davis, I. W., et al.,Nucleic Acids Res, 32: W615-9, 2004).

The Epitopes and the Paratopes

The residues involved in binding between the TLR3 ECD and the three Fabsare shown in FIG. 28B. Fab 12QVQ/QSV bound near the N-terminus of theTLR3 ECD. The conformational epitope was composed of residues from theTLR3 LRRs 3-7 (amino acids 100-221 of SEQ ID NO: 2). The binding of Fab12QVQ/QSV buried approximately 928 Å² and 896 Å² on the antigen andantibody, respectively. For Fab 12QVQ/QSV, the crystal structureidentified following TLR3 (SEQ ID NO: 2) epitope residues: 5115, D116,K117, A120, K139, N140, N141, V144, K145, T166, Q167, V168, S188, E189,D192, A195, and A219. For Fab 12QVQ/QSV, the crystal structureidentified following paratope residues: light chain (SEQ ID NO: 211):G28, S29, Y30, Y31, E49, D50, Y90, D91, and D92. Heavy chain (SEQ ID NO:214): N32, Q54, R56, S57, K58, Y60, Y104, P105, F106, and Y107.

Fab 15EVQ and Fab c1068 bound non-overlapping epitopes spanning LRRs15-23 (amino acids 406-635 of SEQ ID NO: 2) near the C-terminus (FIGS.28A and 28B). Fab 15EVQ buried 1080 Å² and 1064 Å² on the antigen andantibody, respectively, whereas Fab c1068 buried 963 Å² and 914 Å² onthe antigen and antibody, respectively. The epitope for Fab 15EVQ coversresidues K416, K418, L440, N441, E442, Y465, N466, K467, Y468, R488,R489, A491, K493, N515, N516, N517, H539, N541, 5571, L595 and K619 ofTLR3 shown in SEQ ID NO: 2. For Fab 15EVQ, the crystal structureidentified following paratope residues: light chain (SEQ ID NO: 41):Q27, Y32, N92, T93, L94, and S95. Heavy chain (SEQ ID NO: 216): W33,F50, D52, D55, Y57, N59, P62, E99, Y101, Y104, and D106.

For Fab c1068, the crystal structure identified following epitoperesidues on TLR3 (SEQ ID NO: 2): E446, T448, Q450, R453, R473, N474,A477, L478, P480, 5498, P499, Q503, P504, R507, D523, D524, E527, E530,and K559. For Fab c1068, the crystal structure identified followingparatope residues light chain: H30, N31, Y32, N50, E66, S67, G68 (glyc).Heavy chain: T30, T31, Y32, W33, H35, E50, N52, N54, N55, R57, N59, V99,M102, I103, and T104.

Mechanisms of Neutralization and Implication for TLR3 Function

mAb 15EVQ:

The mAb 15EVQ epitope contains TLR3 residues N517, H539 and N541, whichoverlap with the C-terminal dsRNA binding site (Bell et al., Proc. Natl.Acad. Sci. USA, 103:8792-7, 2006). Thus, by not wishing to be bound byany particular theory, it is believed that the mAb 15EVQ competes forTLR3 binding against its ligand and prevents ligand-induced receptordimerization, which is required for the formation of the signaling unit(Liu et al., Science 320:379-81, 2008). FIGS. 29A and 29B illustratesthis direct competition mechanism for mAb 15EVQ. Depending upon theantibody concentration, this mechanism would lead to total inhibition ofpoly(I:C) or dsRNA induced TLR3 activation.

mAb 12QVQ/QSV and mAb c1068:

As shown in FIG. 30, these two antibodies do not have direct clasheswith the dsRNA ligand. Thus, it is unlikely that they would neutralizeTLR3 function in a similar mechanism to that of mAb 15EVQ. The Fabfragments are also oriented away from the ligand (FIGS. 30A, 30B and30C). Structurally, both mAb 12QVQ/QSV and Fab c1068 can bind to asignaling unit (SU) without disrupting its function. Sterically, it isunlikely that the two Fab fragments of a mAb molecule would be able tobind simultaneously the two TLR3 molecules in one SU, and thus preventdsRNA mediated TLR3 dimerization. By not wishing to be bound by anyparticular theory, it is believed that binding of mAb 12QVQ/QSV or mAbc1068 to TLR3 prevents clustering of the signaling unit due to stericclashes between the antibodies and neighboring signaling units. Bindingof TLR3 to dsRNA is not limited to the signaling unit defined by thedsRNA:TLR3 complex (Liu, et al., Science, 320: 379-81, 2008). It ispossible that clustering of multiple SUs can lead to enhancement ofsignaling or that efficient TLR3 signaling requires this clustering. Thepositioning of mAb 12QVQ/QSV and mAb c1068 can block the clustering andresult in neutralization of TLR3 activity. The maximal neutralizationeffects of antibodies would therefore be dependent upon the degree ofseparation of SUs due to antibody binding. As illustrated in FIGS. 30A,30B and 30C, mAb 12QVQ/QSV would cause larger separation than mAb c1068,and this could translate to greater potency of mAb 12QVQ/QSV. This isconsistent with observations that mAb c1068 and mAb 15EVQ can lead to˜50% and 100% TLR3 neutralization at saturation concentrations,respectively, and mAb 12QVQ/QSV exhibits intermediate activity. Thus,combined structural and TLR3 naturalization studies suggest a TLR3signaling model in which the dsRNA:TLR3 signaling units cluster toachieve efficient signaling. mAb 12QVQ/QSV and mAb c1068 and also definea class of antibodies that can partially modulate TLR3 signaling withoutinterfering with ligand binding or receptor dimerization.

1. A method of treating or preventing viral infections comprisingadministering a therapeutically effective amount of an isolated antibodyor fragment thereof to a patient in need thereof for a time sufficientto treat or prevent viral infections, wherein the antibody bindstoll-like receptor 3 (TLR3) amino acid residues K416, K418, L440, N441,E442, Y465, N466, K467, Y468, R488, R489, A491, K493, N515, N516, N517,H539, N541, 5571, L595, and K619 of SEQ ID NO:
 2. 2. A method oftreating or preventing viral infections comprising administering atherapeutically effective amount of an isolated antibody or fragmentthereof to a patient in need thereof for a time sufficient to treat orprevent viral infections, wherein the isolated antibody comprises aheavy chain variable region and a light chain variable region orfragment thereof and the isolated antibody heavy chain variable regionChothia residues W33, F50, D52, D54, Y56, N58, P61, E95, Y97, Y100, andD100b and the isolated antibody light chain variable region Chothiaresidues Q27, Y32, N92, T93, L94, and S95 bind toll-like receptor 3(TLR3) that has the amino acid sequence shown in SEQ ID NO:
 2. 3. Themethod of claim 1, wherein the viral infection is influenza A virusinfection.
 4. The method of claim 2, wherein the viral infection isinfluenza A virus infection.